Steroid 5α-reductase converts testosterone to the more potent androgen, dihydrotestosterone. The molecular mechanisms responsible for maintaining high concentrations of the 5α-reductase type 2 mRNA in the caput epididymidis and for regulating its region-specific expression are unknown. To gain insight into its transcriptional regulation, the cloning and characterization of the 5′ upstream region of 5α-reductase type 2 were undertaken. Sequential deletion analysis was done to map the 2243-base pair (bp) cloned 5′ upstream region, and the constructs were transfected into epididymal PC1 cells and prostatic PC3 cells. In both cell lines, regulatory elements and the minimal promoter were mapped to the 485-bp region upstream of the start codon. Primer extension and 5′ RACE identified one transcriptional start site at 33-bp upstream of the start codon. Using electrophoretic mobility shift assay, a specific band was observed in the −68- to −32-bp region in the presence of nuclear extracts. Supershift and mutational studies confirmed the binding of SP1 and, to a lesser extent, SP3 to the two potential SP1 binding sites and the preference of these proteins to one binding site over the other. SP1 and SP3 were both predominantly immunolocalized to the principal cells of the epididymis and follow distinct distribution patterns in this tissue. These results provide a framework crucial in the further investigation of the transcriptional regulation of 5α-reductase type 2 in the rat epididymis.