Thyroid hormone (TH) has a profound effect on astrocyte differentiation and maturation. Astrocytes cultured under TH-deficient conditions fail to transform from flat polygonal morphology to mature, process-bearing, stellate cells. Supplementation of physiological concentrations of TH initiate gradual transformation of the cells and the process takes ≈ 48 h to complete. The signal transduction pathways associated with TH-mediated maturation of astrocytes have been investigated. TH treatment caused an initial activation of protein kinase A (PKA), with a peak activity at 2 h which fell back to basal level there after. Although there was no visible change in morphology of the cells during the observed activation of PKA, it was sufficient to drive the process of transformation to completion, suggesting the involvement of downstream regulators of PKA. PKA inhibitors as well as the MEK inhibitor PD098059 attenuated the TH-induced morphological transformation. Further studies showed that TH treatment resulted in a biphasic response on the cellular phospho-MAP kinase (p-MAPK or p-ERK) level: an initial decline in the p-ERK level followed by an induction at 18–24 h, both of which could be blocked by a PKA inhibitor. Such sustained activation of p-ERK levels by TH at this later stage coincided with initiation of morphological differentiation of the astrocytes and appeared to be critical for the transformation of astrocytes. The nitric oxide synthase (NOS) inhibitor 7-NI inhibited this induction of p-ERK activity. Moreover, the induction was accompanied by a parallel increase in phospho-CREB activity which, however, persisted at the end of the transformation of the astroglial cells.