OBJECTIVE To elucidate locations of cytosolic carbonic anhydrase isoenzyme (CA-I, CA-II, and CA-III)-positive epithelial cells in equine male reproductive organs. METHODS Descriptive and immunohistochemical study. METHODS 4 clinically normal male horses. METHODS The testis (seminiferous tubules, rete tubules), epididymis (initial, middle, and terminal segments), proximal and distal portions of the ductus deferens, ampulla ductus deferentis, seminal vesicle, prostate, and bulbourethral gland were excised from euthanatized horses after administration of an overdose of pentobarbital. The tissue specimens were quickly placed in fixative solution, dehydrated in ethanol, and embedded; then thin sections were cut. For immunohistochemical staining, antibodies against purified equine CA-I, CA-II, and CA-III were raised in rabbits. After examination of the specificity of each antiserum, the monospecific antisera against carbonic anhydrase isoenzymes were used to localize the isoenzymes. RESULTS Specific staining for CA-III was found in the Sertoli and basal cells of the ductus deferens. Most of the testicular and epididymal tissue, as well as ductus deferens, were virtually negative for the enzymes when stained with the antibody to CA-I and CA-II. In the initial segment of the epididymis, a few principal cells had intense cytoplasmic staining with anti-CA-II. In the male accessory glands, CA-I, CA-II, and CA-III were detected in the epithelial cells of the seminal vesicle, prostate, and bulbourethral gland. CONCLUSIONS In the equine male reproductive tract, the bicarbonate in semen originates mainly from accessory reproductive glands. All 3 isoenzymes may have central roles in the regulation of bicarbonate concentration in seminal plasm and, accordingly, regulate seminal plasma pH. Distribution of CA-III in Sertoli and basal cells of the ductus deferens suggests other specialized physiologic roles.