Chlorpyrifos, one of the most widely used pesticides, exhibits greater toxicity during development than in adulthood. We administered chlorpyrifos to neonatal rats in apparently subtoxic doses that caused no mortality and little or no weight deficits and examined developing brain regions (cerebellum, forebrain, brainstem) for signs of interference with cell development. One-day-old rats given 2 mg/kg sc of chlorpyrifos showed significant inhibition of DNA synthesis in all brain regions within 4 hr of treatment; equivalent results were obtained when a small dose (0.6 microgram) was introduced directly into the brain via intracisternal injection, indicating that the actions were not secondary to systemic toxicity. Inhibition of DNA synthesis was also seen at 8 days of age; however, at this point, there was regional selectivity, with sparing of the cerebellum. Between 1 and 8 days of age, brain regions develop wide disparities in cholinergic innervation; accordingly, we tested whether the effect of chlorpyrifos was mediated through cholinergic actions on nicotinic receptors known to mediate inhibition of DNA synthesis. Pretreatment with mecamylamine caused a decline in DNA synthesis by itself, but nevertheless prevented the effect of chlorpyrifos. Additionally, chlorpyrifos administration at 1 day of age caused an even larger inhibition of protein synthesis throughout the brain; the effect was distinct from that on DNA synthesis, as it diminished substantially by 8 days of age and did not develop any regional selectivity. The effects of chlorpyrifos on DNA and protein synthesis were not secondary to generalized cell damage or suppression of cell metabolism, as evidenced by maintenance of normal ornithine decarboxylase activities. These results indicate that low doses of chlorpyrifos target the developing brain during the critical period in which cell division is occurring, effects which may produce eventual cellular, synaptic, and behavioral aberrations after repeated or prolonged subtoxic exposures.