An acidic luminal pH (ref. 1-3) is involved in sperm maturation, and in maintaining sperm in an immotile state in the epididymis and vas deferens (2,4-6). Neutralization by prostatic fluid is one of a complex series of events that triggers sperm motility (2,7,8). Failure of the acidification mechanism might, therefore, result in poor sperm maturation, premature motility and infertility. We have shown that a vacuolar (H+)-ATPase is expressed at high levels on the luminal plasma membrane of specialized cells in the epididymis (9), which closely resemble acid-secreting kidney intercalated cells (10,11). We now show that similar cells are also present in the vas deferens, and that a bafilomycin-sensitive proton flux can be detected using a noninvasive proton-selective vibrating probe. Up to 80% of the net proton secretion in the vas deferens is inhibited by bafilomycin, consistent with a major role of a vacuolar-type (H+)-ATPase in this process. This acidification mechanism is a potential target for novel strategies aimed at modulating the acidification capacity of parts of the male reproductive tract and, therefore, in regulating male fertility.