ABSTRACT: In addition to the maturation of sperm, the epididymis also serves to protect sperm from harmful reactive oxygen species. To this end, various antioxidant enzymes are produced by the epididymis, such as glutathione S-transferases (GSTs), a family of dimeric proteins that catalyze the conjugation of glutathione to various electrophilic compounds, thus providing cellular detoxification. In the present study, the regulation of the Yb1 subunit of GST was examined in Bouin-fixed epididymides of adult control, orchidectomized (O) rats with or without testosterone (T) supplementation and efferent duct—ligated (EDL) rats using light microscope immunocytochemistry with an anti-Yb1—GST antibody. The intensely reactive ciliated cells of the efferent ducts and principal cells of the epididymis showing a checkerboard staining pattern were unaltered in their expression of Yb1-GST after all experimental procedures, suggesting their regulation by factors other than of testicular origin. On the other hand, the intense reaction of narrow/apical cells and moderate reaction of basal cells of the proximal initial segment of control animals became negligible in O rats and was not restored with T supplementation. As staining was also absent after EDL, the data suggest that a luminal testicular factor(s), other than androgens, regulates expression of Yb1-GST in narrow/apical and basal cells of the proximal initial segment. Although basal cells of the caput and cauda epididymidis were unreactive after all experimental protocols, as also noted in controls, the intensely reactive basal cells of the corpus epididymidis of control animals became unreactive in O animals. However, Yb1-GST expression was restored to these cells with T supplementation, and as there was no effect on Yb1-GST expression after EDL, the data suggest that circulating testosterone or one of its metabolites regulates expression of Yb1-GST in basal cells of the corpus region. Taken together, these data indicate a differential regulation with respect to the expression of Yb1-GST in the various cell types and regions of the epididymis.