The lumen of the epididymis is the site where spermatozoa undergo their final maturation and acquire the capacity to become motile. An acidic luminal fluid is required for the maintenance of sperm quiescence and for the prevention of premature activation of acrosomal enzymes during their storage in the cauda epididymis and vas deferens. We have previously demonstrated that a vacuolar H+-ATPase [proton pump (PP)] is present in the apical pole of apical and narrow cells in the caput epididymis and of clear cells in the corpus and cauda epididymis and that this PP is responsible for the majority of proton secretion in the proximal vas deferens. We now show that PP-rich cells in the vas deferens express a high level of carbonic anhydrase type II (CAII) and that acetazolamide markedly inhibits the rate of proton secretion by 46.2 +/- 6.1%. The rate of acidification was independent of Cl- and was strongly inhibited by SITS under both normal and Cl--free conditions (50.6 +/- 5.0 and 57. 5 +/- 6.0%, respectively). In the presence of Cl-, diphenylamine-2-carboxylate (DPC) had no effect, whereas SITS inhibited proton secretion by 63.7 +/- 11.3% when applied together with DPC. In Cl--free solution, DPC markedly inhibited proton efflux by 45.1 +/- 7.6%, SITS produced an additional inhibition of 18.2 +/- 6.6%, and bafilomycin had no additive effect. In conclusion, we propose that CAII plays a major role in proton secretion by the proximal vas deferens. Acidification does not require the presence of Cl-, but DPC-sensitive Cl- channels might contribute to basolateral extrusion of HCO-3 under Cl--free conditions. The inhibition by SITS observed under both normal and Cl--free conditions indicates that a Cl-/HCO-3 exchanger is not involved and that an alternative HCO-3 transporter participates in proton secretion in the proximal vas deferens.