The epididymal epithelium contributes to formation of a luminal fluid that is essential for the protection of spermatozoa from a variety of insults including changes in oxygen tension. A key regulator of the response to oxygen debt in many cells is hypoxia-inducible factor-1 (HIF-1). A transcription factor composed of α and β subunits, HIF-1 activates genes that mediate oxygen homeostasis and cell survival pathways or trigger cell death responses. Previously we have shown that HIF-1α mRNA is expressed in the adult rat epididymis. Goals of this study were to determine whether HIF-1α protein is activated by ischemia in the rat epididymis, to determine whether epididymal HIF-1α mRNA expression is androgen dependent, and to identify epididymal cell types expressing HIF-1α and β. Immunoblot analysis revealed that HIF-1α protein is primarily present in corpus and cauda of the normoxic epididymis and unaffected by ischemia, whereas HIF-1β was detected equally in all regions and also unaffected by ischemia. HIF-1α mRNA expression in all regions was not affected by 15 days bilateral orchiectomy. Principal cells stained positive for HIF-1α by immunocytochemistry, with the epithelium of initial segment and caput epididymidis staining less intensely than corpus and cauda. HIF-1β immunoreactivity was equally present in principal cells in all regions. Clear, narrow, and basal cells were unreactive for HIF-1α and β. The presence of HIF-1 in normoxic epididymis and the regional distribution of HIF-1α suggests fundamental differences in how proximal and distal regions of the epididymis maintain oxygen homeostasis to protect the epithelium and spermatozoa from hypoxia.