In rats, an acidic luminal pH maintains sperm quiescence during storage in the epididymis. We recently showed that vacuolar H ATPase-rich cells in the epididymis and vas deferens are involved in the acidification of these segments. Treatment of rats with cadmium (Cd) leads to alkalinization of this fluid by an unknown mechanism. Because Cd may affect H ATPase function, we examined 1) the in vivo effect of Cd poisoning on H ATPase-rich cell morphology and on the abundance and distribution of the 31-kDa H ATPase subunit in cells along the rat epididymis, and 2) the in vitro effect of Cd on H ATPase activity and function in the isolated vas deferens. Immunofluorescence and immunoblotting data from rats treated with Cd for 14–15 days (2 mg Cd/kg body mass/day) showed that 1) H ATPase-positive cells regressed to a prepubertal phenotype, and 2) H ATPase was lost from the apical pole of the cell and was redistributed into an intracellular compartment. In experiments in vitro, Cd inhibited bafilomycin-sensitive ATPase activity in isolated total cell membranes and, as measured using a proton-selective extracellular microelectrode, inhibited proton secretion in isolated vas deferens. We conclude that alkalinization of the tubule fluid in the epididymis and vas deferens of Cd-treated rats may result from the loss of functional H ATPase enzyme in the cell apical domain as well as from a direct inhibition of H ATPase function by Cd.