Pod1 is a member of the basic helix-loop-helix (bHLH) family of transcription factors that have been implicated in the regulation of sexual differentiation and gonadal development in mammals. However, to date, little is known about the role of Pod1 in nonmammalian vertebrate gonadogenesis. We cloned and characterized the Pod1 gene from tilapia. The tilapia Pod1 gene contains an open reading frame (ORF) of 525 nucleotides which potentially codes for a protein with 174 amino acids. Sequence alignment revealed that the deduced tilapia protein sequence shared high homology (79.5% to 90.5%) with the Pod1 sequences of other vertebrates. The tissue distribution of Pod1 revealed by RT-PCR showed that it had varied expression patterns in adult tilapia. In situ hybridization was performed to examine the temporal and spatial expression patterns of Pod1 during tilapia sexual differentiation and gonadal development. In the undifferentiated gonad, Pod1 was expressed in the somatic cells of both sexes. Subsequently, Pod1 expression in tilapia persisted in differentiated juvenile and adult ovary and testis. Our data indicate for the first time that Pod1 is not only necessary for the onset of sexual differentiation, but also plays an important role in gonadal development in the teleost.