Recently, it has been shown that the prenatal vitamin D(3) depletion is associated with altered brain development. Given the antiproliferative and pro-apoptotic properties of vitamin D(3) in various cell types, we examined the effects of maternal vitamin D(3) deprivation on cell proliferation and apoptosis within the rat cortex at several developmental stages. Our results confirm that vitamin D(3) regulates these processes in the developing brain at both cellular and molecular levels. Compared to control animals, the embryos and pups from vitamin D(3) depleted mothers had significantly less apoptotic cells, this finding being most pronounced at birth. Additionally, there were significantly more mitotic cells but this was not associated with any particular developmental period. Targeted gene arrays specific for apoptosis and cell cycle genes confirmed a pattern of transcription deregulation in the deplete group consistent with the known properties of vitamin D(3). While most current vitamin D(3) research is focussed on the pro-apoptotic and prodifferentiating properties of vitamin D(3) as adjuncts for the treatment of cancers, our findings highlight the important role that this hormone plays in normal development via these same properties specifically in the brain.