Recently, a large cluster of homeobox genes was discovered on the X chromosome that is expressed in reproductive tissues after birth. It is postulated that these reproductive homeobox genes on the X chromosome (Rhox) encode transcription factors that regulate gametogenesis. In support of this, male mice lacking the founding member of this gene cluster, Rhox5, are subfertile, exhibiting increased germ-cell apoptosis and a defect in sperm motility. To identify RHOX5 targets, microarray analyses were used to identify genes differently expressed in postnatal testes from Rhox5-null and control littermates. Highly overrepresented were genes that encode proteins involved in cellular metabolism. Several lines of evidence indicated that one of these, insulin II, is a direct target of RHOX5. Microarray analysis was also used to identify genes differentially expressed in response to physiological levels of Rhox5 in a Sertoli-cell line. Among the few genes identified, the netrin-1 receptor UNC5c, a proapoptotic molecule that is inhibited by RHOX5, was also regulated in vivo, and is thus a candidate to be downstream of RHOX5 in a prosurvival germ-cell pathway. To understand the means by which Rhox5 expression is restricted to Sertoli nurse cells in the testis, a variety of molecular approaches were used in both Sertoli-cell lines and mice. This analysis revealed that both positive and negative cis elements collaborate to confer Sertoli cell-specific gene expression. Acting on the positive cis elements are androgen receptor and GATA transcription factors. Collectively, the results of this study provide an initial glimpse into the regulatory networks that control spermatogenesis.