The major regulators of synaptic glutamate in the cerebral cortex are the excitatory amino acid transporters 1–3 (EAAT1–3). In this study, we determined the cellular and temporal expression of EAAT1–3 in the developing human cerebral cortex. We applied single- and double-label immunocytochemistry to normative frontal or parietal (associative) cortex samples from 14 cases ranging in age from 23 gestational weeks to 2.5 postnatal years. The most striking finding was the transient expression of EAAT2 in layer V pyramidal neuronal cell bodies up until 8 postnatal months prior to its expression in protoplasmic astrocytes at 41 postconceptional weeks onward. EAAT2 was also expressed in neurons in layer I (presumed Cajal–Retzius cells), and white matter (interstitial) neurons. This expression in neurons in the developing human cortex contrasts with findings by others of transient expression exclusively in axon tracts in the developing sheep and rodent brain. With western blotting, we found that EAAT2 was expressed as a single band until 2 postnatal months, after which it was expressed as two bands. The expression of EAAT2 in pyramidal neurons during human brain development may contribute to cortical vulnerability to excitotoxicity during the critical period for perinatal hypoxic–ischemic encephalopathy. In addition, by studying the expression of EAAT1 and EAAT2 glutamate transporters, it was possible to document the development of protoplasmic astrocytes. J. Comp. Neurol. 520:3912–3932, 2012. © 2012 Wiley Periodicals, Inc.