Pit-1 is a POU-domain transcription factor that promotes growth hormone (GH), prolactin (PRL), and thyroid-stimulating hormone β subunit (TSHβ) gene expression in the pituitary gland. Alternative splicing of Pit-1 gene transcripts has been shown to give rise to several variants with discrete transactivation properties. Recently, we identified a mouse Pit-1 w that is generated by alternative promoter usage and is expressed in a variety of tissues including the testis. Using a combination of reverse-transcription polymerase chain reaction analyses and luciferase reporter gene assays, we investigated the possible role of Pit-1 w in the mouse testis. In postnatal testicular development, the expression of Pit-1 w mRNA was significantly up-regulated between 18 and 20 days after birth when the numbers of secondary spermatocytes and spermatids have been reported to increase in mice. The PRL mRNA, but not the mRNAs for GH or TSHβ, showed intratesticular expression patterns that were similar to those of the Pit-1 w mRNA. In experimental unilaterally cryptorchid testes of adult mice, spermatid numbers were extremely low and the expression levels of both the Pit-1 w and PRL mRNAs dropped dramatically. Furthermore, in the luciferase reporter gene assays, we found that Pit-1 w specifically transactivated the PRL promoter but had no effect on the promoters of GH or TSHβ. These results suggested that Pit-1 w could be involved in the paracrine/autocrine system in mice and may be necessary for normal testicular function via its possible role in regulating PRL expression in testicular germ cells. This is the first report demonstrating the possible role of Pit-1 w in mammals.