Several genes expressed in the initial segment of the epididymis depend on factors from the testis that reach the epididymis via the luminal system. These include gamma-glutamyl transpeptidase mRNA IV (Ggt_pr4), steroid 5 alpha reductase (Srd5a1), glutathione peroxidase 5 (Gpx5), and cystatin-related epididymal spermatogenic (Cst8) genes. Promoter analyses indicated that these genes contain several ETS DNA-binding sites. Members of the polyomavirus enhancer activator 3 (ETV4) family bind to ETS sites on the promoter of target genes to regulate transcription. In this study, the role of ETV4 family members (ETV4, ETV5, ETV1) in the transcription of initial segment specific genes was evaluated. All three ETV4 family mRNAs are expressed in the principal cells of the initial segment and depend upon the presence of testicular luminal fluid factors. ETV4 protein was localized to principal cell nuclei and displayed the highest expression in the most proximal region of the initial segment. In addition, ETV4 protein levels were diminished after loss of testicular luminal fluid factors. A dominant-negative construct of ETV5 was in vivo electroporated into the initial segment to determine if ETV4 family members can regulate the transcription of testicular luminal fluid factor-regulated genes. Quantitative PCR indicated that 1 day postelectroporation, all three ETV4 family member mRNAs were significantly decreased. In addition, Ggt_pr4, Srd5a1, and Gpx5 mRNA levels were also significantly decreased. The data suggest that ETV4 family members regulate their own expression, and that they regulate transcription of a subset of genes that are dependent upon testicular luminal fluid factors.