Sex-reversed (Sxr) is a duplication of the sex-determining region of the Y chromosome, which gets transposed to a paternal X chromosome. Chromosomally female (XX) zygotes that receive this XSxr chromosome develop as apparent males. Previous work on XXSxr mice (called pseudomales) showed extracellular matrix (ECM) ultrastructural abnormalities in the epididymis and testis. This study examined the biochemical nature of these abnormalities. More hydroxyproline (an indicator of collagen) was noted in the pseudomale testis and epididymis compared to normal male tissues. Western blot analysis showed increased collagen IV in the pseudomale testis and epididymis. In both the hydroxyproline and collagen IV studies, the epididymis was found to contain higher levels of these substances than the testis for both genotypes. There also appeared to be increased messenger RNA for tissue inhibitor of metalloproteinases (Timp), a regulator of collagen, in the pseudomale testis. Data from these studies seem to indicate that the XXSxr genotype influences ECM deposition and/or turnover and exerts a direct genetic influence on the development of the testis and epididymis. According to the existing paradigm of mammalian sexual development, the epididymis is expected to be normal in the presence of adequate androgenization and independent of chromosomal and genetic sex. The results presented here differ from what would be predicted by this paradigm.