The organophosphate pesticide, chlorpyrifos (CPF), is a developmental neurotoxicant. In cell cultures, CPF affects gliotypic cells to a greater extent than neuronotypic cells, suggesting that glial development is a specific target. We administered CPF to developing rats and examined the levels of glial fibrillary acidic protein (GFAP), an astrocytic marker. Prenatal CPF exposure (gestational days 17-20) elicited an increase in GFAP levels in fetal brain, but the effect was seen only at high doses that elicited maternal and fetal systemic toxicity. Early postnatal (PN) CPF treatment (PN1-4) elicited effects only in the cerebellum of male rats; GFAP was suppressed initially (PN5) and showed a rebound elevation (PN10) before returning to normal values by PN30. In contrast, when we administered CPF during the peak of gliogenesis and glial cell differentiation (PN11-14), GFAP was initially decreased across all brain regions and in both sexes; in males, subsequent elevations were seen on PN30, with the largest effect in the striatum; females also showed an increase in striatal GFAP. Our results indicate that CPF disrupts the pattern of glial development in vivo, with the maximum effect corresponding to the peak period of gliogenesis and glial cell differentiation. As glia are responsible for axonal guidance, synaptogenesis and neuronal nutrition, glial targeting suggests that these late-occurring developmental processes are vulnerable to CPF, extending the critical period for susceptibility into stages of synaptic plasticity, myelination, and architectural modeling of the developing brain.