Animal studies indicate neurobehavioral anomalies after prenatal exposure to benzo[a]pyrene (BaP). In order to determine if BaP directly affects neurodevelopment, we compared its effects to those of the organophosphate insecticide, chlorpyrifos (CPF), in undifferentiated and differentiating neuronotypic PC12 cells, evaluating indices of cell replication, cell number, neurite outgrowth and phenotypic differentiation. Unlike CPF, BaP did not inhibit DNA synthesis in undifferentiated cells. In cells undergoing nerve growth factor-induced differentiation, CPF reduced cell numbers (assessed by DNA content) whereas BaP increased them, suggesting a delay in the transition between cell replication and differentiation. Indices of cell enlargement (total protein/DNA) and neurite outgrowth (membrane protein/DNA) also showed opposite effects of CPF (increases) and BaP (decreases). We directly confirmed BaP impairment of neurodifferentiation by measuring markers for the two neurotransmitter phenotypes expressed by PC12 cells: tyrosine hydroxylase (dopamine phenotype) and choline acetyltransferase (acetylcholine phenotype). BaP significantly reduced both markers in differentiating cells, with a preferentially greater effect on the acetylcholine phenotype. Our results indicate that low, non-toxic levels of BaP can impair neurodifferentiation, resulting in excess cell numbers at the expense of the emergence of neurotransmitter phenotypes. BaP thus has direct actions on developing neuronal cells that could contribute to the adverse neurodevelopmental effects seen with in vivo exposures.