The vacuolar H+ ATPase is inhibited by N-ethylmaleimide (NEM), a sulfhydryl compound, suggesting the involvement of a sulfhydryl group in this transport process. We have examined the effects of several sulfhydryl-containing compounds on the vacuolar H+. ATPase of rabbit renal cortical endosomes. A number of such compounds were effective inhibitors of endosomal H+ transport at 10(-5)-10(-6) M, including NEM, mersalyl, aldrithiol, 5,5' dithiobis (2-nitrobenzoic acid), p-chloromercuribenzoic acid (PCMB) and p-chloromercuriphenyl sulfonic acid (PCMBS). NEM, mersalyl, aldrithiol and PCMBS had no effect on pH-gradient dissipation, whereas PCMB decreased the pH gradient faster than control. In the absence of ATP, PCMB (10(-4) M) stimulated endosomal 36Cl- uptake, particularly in the presence of an inside-alkaline pH gradient (pHin = 7.6/pHout = 5.5). This result was not an effect of PCMB on the Cl(-)-conductive pathway. The less permeable PCMBS did not stimulate 36Cl- uptake. The effects of PCMB were concentration dependent and were prevented by dithioerithritol. ATP-dependent 36Cl- uptake was decreased by addition of PCMB. Finally, PCMB had no effect on 45Ca2+ uptake. These results support the presence of two functionally important sulfhydryl groups in this endosomal preparation. One such group is involved with ATP-driven H+ transport and must be located on the cytoplasmic surface of the endosomal membrane. The second sulfhydryl group must reside on the internal surface of the endosomal membrane and relates to a PCMB-activated Cl-/OH- exchanger that is functional both in the presence and absence of ATP. This endosomal transporter is similar to the PCMB-activated Cl-/OH- exchanger recently described in rabbit renal brush-border membranes.