Homeobox genes encode DNA-binding proteins that regulate the transcription of subordinate downstream genes. In this study, we show that the Pem homeobox gene is expressed and regulated in a unique manner in neonatal and adult rats. Pem gene expression was primarily confined to reproductive tissue: epididymis, testis, ovary, and placenta. In the epididymis, Pem transcripts were localized by in situ hybridization analysis to the proximal cauda region, a site where spermatozoa gain fertilization competence. Pem mRNA levels dramatically increased between Days 21 and 26 postpartum in the epididymis, coincident with the induction of genes known to be responsive to testosterone (T), but in contrast to that of other genes examined, including the Hoxc-8 homeobox gene. Pem expression was shown to be T-dependent on the basis of an absence of Pem transcripts in the epididymides of hypophysectomized rats and restoration of normal Pem mRNA levels after administration of T. In the testis, Pem mRNA levels were elevated earlier (between Days 12 and 15 postpartum) and less dramatically than in epididymis. Pem gene expression in the testis was depressed after hypophysectomy, but normal levels of Pem expression were not restored by T treatment under the same conditions that permitted normal Pem expression in the epididymis. To our knowledge Pem is the first reported putative transcription factor that has been demonstrated to depend on androgens for expression in the epididymis, and thus Pem is a candidate as a regulator of androgen-dependent events in this tissue.