Fetal or neonatal chlorpyrifos (CPF) exposure affects serotonin (5HT) synaptic function and related behaviors in adulthood. We examined the critical period and dose threshold for effects on 5HT and assessed their emergence in adolescence. Pregnant rats were given CPF (1 or 5 mg/kg/day) from gestational days (GD)17–20 or GD9–12 and 5HT levels and turnover were evaluated on postnatal day 30; the lower dose lies below the threshold for inhibition of fetal brain cholinesterase. GD17–20 exposure increased 5HT turnover in brain regions containing either 5HT projections or cell bodies, with a preferential effect in males. Shifting the exposure to GD9–12 also augmented 5HT activity but only in the cerebral cortex and without sex preference. Similar, but lesser effects were seen for dopamine turnover in the same regions. These results indicate that, in a critical developmental period, apparently subtoxic exposures to CPF produce long-term activation of 5HT systems.