Neurogenesis begins with the closure of the neural tube around mid gestation and continues in the rat for about two weeks postnatally. Therefore, we investigated the role of neuronatin, a novel cDNA that we cloned from neonatal rat brain (Joseph et al., Biochem. Biophys. Res. Commun., 201 (1994) 1227–1234), in brain development. Further studies described in the present manuscript, lead to the identification of two alternatively spliced forms of neuronatin mRNA, α and β, with the same open reading frame. Neuronatin-α encoded a novel protein of 81 aa, and the β-form encoded 54 aa. Both forms were identical, except that the α-form had an additional 81 bp sequence inserted into the middle of the coding region. On Northern analyses, neuronatin mRNA was relatively selective for the brain. It first appeared at E11–14, a time when the neural tube has closed and neuroepithelial proliferation initiated, became pronounced at E16–19 with a surge in neurogenesis, and declined postnatally to adult levels with the completion of neurogenesis. In order to determine whether there were other forms of neuronatin mRNA, and to study the expression of the α and β forms separately during development, reverse transcriptase-polymerase chain reaction was carried out using primers flanking the coding region of the α and β forms. The RT-PCR results clearly indicated that there were only two forms of neuronatin. The β-forms first appeared at E11–14, whereas the α-form was present even earlier at E7–10. Together, these findings indicate that the two forms of neuronatin mRNA are regulated differently during brain development. The appearance of the β-form at mid-gestation, coinciding with the onset of neurogenesis, may suggest that alternative splicing of neuronatin mRNA has relevance in brain development.