The catalytic domains of PTP1B and TCPTP share a high degree of primary (72% identity and 86% similarity; TCPTP residues 43–288) and tertiary structure similarity and have similar active sites [36,113] (Fig. 1). PTP1B and TCPTP are among the most closely related of the classical phosphotyrosine-specific PTPs, with members of this enzyme subfamily in general sharing an approximate 35% sequence identity [36,113]. In particular, both PTPs share a second ‘phosphotyrosine-binding pocket’ that allows for the selective recognition of substrates phosphorylated on tandem tyrosines [94,114,115], such as the IR β-subunit activation loop Y1162/Y1163 phosphorylation site [92,96,114] (Fig. 2), or the JAK1/2/3 and TYK2 activation loop (Y1022/Y1023 in JAK1; Y1007/Y1008 in JAK2; Y980/Y981 in JAK3; Y1054/Y1055) phosphorylation sites [56,106,116]. Despite their similarity, TCPTP and PTP1B exhibit a high degree of substrate selectivity in a cellular context. This substrate selectivity is evident in the distinct phenotypes of Ptpn1−/− versus Ptpn2−/− mice [34,35,110] and substantiated by extensive molecular and substrate identification studies. For example, PTP1B can dephosphorylate JAK2, but not JAK1/3, whereas TCPTP dephosphorylates JAK1/3, but not JAK2 [56,106,116]. Moreover, PTP1B and TCPTP dephosphorylate distinct platelet-derived growth factor receptor tyrosine phosphorylation sites to differentially control signaling and cell migration/chemotaxis . Their unique substrate selectivities are highlighted in particular by their opposing roles in the regulation of Src family PTKs. PTP1B dephosphorylates the c-Src C-terminal Y529 (Y527 avian) inhibitory phosphorylation site to activate c-Src [117,118], whereas TCPTP dephosphorylates the c-Src Y418 (Y416 avian) PTK activation loop site to inactivate c-Src . PTP1B’s ability to dephosphorylate the c-Src Y529 site is dependent on a noncatalytic C-terminal domain proline-rich sequence that can interact with proteins containing N-terminal Src homology 3 domains [119–122]; this proline-rich sequence is not present in TCPTP (Fig. 1). Their differential contributions to SFK regulation is evident in TNF signaling, where PTP1B promotes and TCPTP attenuates TNF-induced and mitogen-activated protein kinase-mediated inflammatory responses [102,123,124], and in pancreatic β cells, where high-fat-diet-induced increases in PTP1B and decreases in TCPTP serve to promote SFK activation and attenuate ER stress . Furthermore, PTP1B and TCPTP function cooperatively to regulate the intensity and duration of IR activation and signaling by coordinately regulating Y1162/Y1163 phosphorylation [92,96,126] and also work in concert to regulate MET receptor phosphorylation . PTP1B and TCPTP also promote distinct effects on signaling by virtue of their different subcellular locations . For example, both PTP1B and 45 kDa TCPTP can directly regulate STAT6 Y641 phosphorylation, but exert their effects in the cytoplasm and nucleus respectively [127,128]. Importantly, recent studies have highlighted that their substrate specificity, selectivity and cooperativity extend in vivo in the central control of body weight and glucose homeostasis, where PTP1B attenuates hypothalamic leptin signaling at the level of JAK2 (but not STAT3 or the leptin receptor ) in the cytoplasm, and TCPTP dephosphorylates STAT3 in the nucleus [56,103,106,116] (Fig. 2). These studies have shown that conditional deletion of PTP1B and TCPTP in neuronal and glial cells has additive effects in the promotion of central leptin sensitivity and in the attenuation of high-fat-diet-induced obesity and the concomitant development of insulin resistance and glucose intolerance . These studies have served to highlight the nonredundant and essential nature of even highly related phosphatases in vivo. Moreover, these studies have underscored the potential of combinatorially inhibiting PTP1B and TCPTP in the hypothalamus for the prevention of obesity and the improvement of whole-body glucose homeostasis.