Developmental neurotoxicity (DNT) is an important issue in children's health. Neurogenesis occurs throughout the early fetal to the postnatal period. The proliferation of embryonic stem cells can be a target for toxicants, especially genotoxic compounds. 5-Bromo-2'-deoxyuridine (BrdU), a thymidine analogue, has been used as a marker for proliferating cells. However, we reported that prenatal BrdU exposure induced behavioral abnormalities such as hyperactivity in rat and mouse offspring. In this study, to further clarify the toxic effect of BrdU on the early neurogenesis and to examine the usefulness of the evaluation of this process in DNT, C57BL/6 mice were exposed to 100 mg/kg of BrdU once on gestational day (GD) 9 or 11, and serial sections from a wide variety of areas of the embryonic brains 24 h after the exposure were examined. BrdU exposure on GD11 induced cell death in some specific areas, such as the neocortex and striatum, but not in the substantia nigra, raphe and pons, even though BrdU was incorporated into those cells. BrdU decreased the number of cells positive for phosphorylated histone 3 (phospho-histone 3), a marker for proliferating cells at metaphase of mitosis, in the cortex, mammillary body and cerebellum, suggesting that BrdU affected the proliferation of neural stem cells. Exposure on GD9 did not induce cell death in the fetal brain. These results indicate that BrdU actually impaired the early neurogenesis, supporting the postnatal results, and demonstrated that embryonic neurogenesis has heterogeneous sensitivity to the genotoxic agents BrdU that differs according to the area and developmental stage. The evaluation of events in early neurogenesis such as the proliferation of neural stem cells shortly after chemical exposure will be one of the valuable endpoints for studying postnatal neurodevelopmental disorders.