Chlorpyrifos is used increasingly as a pesticide in place of more toxic alternatives such as parathion. Although chlorpyrifos is not a potent dysmorphogen, recent reports that fetal or infant exposures may exceed acceptable limits have raised concern about the potentially more subtle effects on brain development. In the current study, whole rat embryo culture was used to study the effects of chlorpyrifos at the neural tube stage of development. On embryonic day 9.5, embryos were exposed to 0.5, 5, or 50 micrograms/ml of chlorpyrifos. After 48 hr (embryonic day 11.5), embryos were examined for dysmorphogenesis and were then processed for light microscopic examination of the neuroepithelium. Examination of 1-micron-thick toluidine blue-stained sections of the forebrain and hindbrain region revealed reduced and altered mitotic figures, with dispersion and disorientation of the mitotic layer. In addition, cytotoxicity was evidenced by cytoplasmic vacuolation, enlargement of intercellular spaces, and the presence of a significant number of apoptotic cells. These alterations were evident even at the lowest concentration of chlorpyrifos, which produced no dysmorphogenesis. The effects were intensified at higher concentrations, which were just at the threshold for dysmorphogenesis; the neuroepithelial abnormalities, however, were still present in embryos that were not dysmorphogenic. Our results in rat embryo culture support the idea that chlorpyrifos specifically targets brain development at low concentrations, indicating the need to reevaluate the safety of this compound for exposure in vivo.