DAX-1 (DSS-AHC Critical Region on the X Chromosome-1) is a member of the nuclear hormone receptor superfamily that has an important role in steroidogenesis and gonadogenesis. To investigate the role of DAX-1 in the testis, a yeast two-hybrid screen was performed and SOX6, member of the Sry box (SOX) protein family, was cloned as candidate. The interaction was confirmed biochemically and expression of SOX6 overlapped with that of DAX-1 in the developing gonad, as well as in Sertoli cells of the adult testis. We show here that DAX-1 is able to inhibit splicing in in vivo and in vitro splicing assays, and this inhibition is relieved by the addition of SOX6. SOX6 appears to inhibit the interaction between DAX-1 and the splicing machinery, thus providing a likely mechanism for functional interference. We conclude that DAX-1 and SOX6 proteins interact, have overlapped expression in the testis, and act antagonistically during pre-mRNA splicing. Developmental Dynamics 238:1595-1604, 2009. (c) 2009 Wiley-Liss, Inc.