The widely used organophosphate insecticide, chlorpyrifos (CPF), elicits neurobehavioral abnormalities after apparently subtoxic neonatal exposures. In the current study, we administered 1 or 5 mg/kg/day of CPF to pregnant rats on gestational days 9-12, the embryonic phase spanning formation and closure of the neural tube. Although there were no effects on growth or viability, offspring showed behavioral abnormalities when tested in adolescence and adulthood. In the CPF-exposed groups, locomotor hyperactivity was noted in early T-maze trials, and in the elevated plus-maze; alterations in the rate of habituation were also identified. Learning and memory were adversely affected, as assessed using the 16-arm radial maze. Although all CPF-exposed animals eventually learned the task, reference and working memory were impaired in the early training sessions. After training, rats in the CPF group did not show the characteristic amnestic effect of scopolamine, a muscarinic acetylcholine antagonist, suggesting that, unlike the situation in the control group, muscarinic pathways were not used to solve the maze. These results indicate that apparently subtoxic CPF exposure during neurulation adversely affects brain development, leading to behavioral anomalies that selectively include impairment of cholinergic circuits used in learning and memory. The resemblance of these findings to those of late gestational or neonatal CPF exposure indicates a prolonged window of vulnerability of brain development to CPF.