Acidic epididymal fluid mainly accounts for sperm quiescence during storage in the epididymis. Carbonic anhydrase (CA) is an enzyme involved in proton and bicarbonate secretion in various epithelia. Therefore, we elucidated the distribution of the cytoplasmic (CA II) and membrane-associated (CA IV) isoenzymes in rat epididymis using polyclonal rabbit antisera to these isoenzymes in conjunction with immunohistochemical and immunoblotting techniques. CA IV was localized in the apical plasma membrane of principal epithelial cells in the distal caput, corpus, and proximal cauda epididymides, the staining intensity being most intense in the corpus segment. The epithelium of the ductus deferens, seminal vesicle, and ventral prostate was devoid of staining. CA II was present in the narrow cells of the initial segment and in the epithelial cells of the distal caput, corpus, and proximal cauda epididymides. Immunoblotting of different epididymal segments for CA IV and II revealed with anti-CA IV serum a distinct 39-kDa polypeptide band in the corpus segment and with anti-CA II serum a 29-kDa polypeptide band in all segments, with the band most intense, however, in the corpus segment. Our results imply that in rat epididymis both bicarbonate reabsorption and proton secretion are involved in epididymal fluid acidification. By analogy with the kidney proximal tubule, we suggest that CA IV is involved in bicarbonate reabsorption mainly occurring in the corpus epididymidis. The presence of CA II in epididymal epithelial cells is probably involved in the supply of protons for secretion mediated by various ion transport mediators.