Neurogenin 3 (Ngn3), a proneural gene controlled by the Notch receptor, is implicated in the control of dendrite morphology and synaptic plasticity of cultured hippocampal neurons. Here we report the localization and subcellular distribution of Ngn3 in the hippocampus in vivo and in neuronal cultures. In situ hybridization showed Ngn3 mRNA expression in the pyramidal layer and dentate gyrus of adult mouse hippocampus. Immunohistochemistry studies revealed that Ngn3 localization is mostly cytoplasmic in the hippocampal eminence at embryonic day (E)17 and postnatal day (P)0. At P10 it is cytoplasmic in CA1-CA3 pyramidal neurons and nuclear in granule cells of the dentate gyrus. In the adult hippocampus Ngn3 is localized in the nucleus and cytoplasm of both pyramidal neurons and granule cells. During development of cultured hippocampal neurons, Ngn3 mRNA expression is higher at stages of neuronal polarization, as judged by reverse-transcription polymerase chain reaction (RT-PCR), and it is mostly cytoplasmic. The tracking of the subcellular localization of Ngn3 in neurons infected with a virus expressing myc-Ngn3 suggests that the protein is quickly translocated to the cell nucleus after synthesis and then reexported to the cytoplasm. Treatment with leptomycin B, a potent and specific inhibitor of the exportin CRM1, induced its accumulation into the nucleus, suggesting that CRM1 mediates the nuclear export of Ngn3. These results suggest that Ngn3 may play a role in neuronal development by actions in the cytoplasm.