A proton pump in diverse biological systems consists of two structural units with separate but integrated functions, the F0-F1-ATPase. We tested by chemical perturbation the possibility that such a proton pump might be involved in urinary acidification conducted by urinary epithelia (UF0-UF1-ATPase). Tyrosine-reactive chemicals and N,N'-dicyclohexylcarbodiimide, known to block the proton channel unit (F0), also inhibited urinary acidification, as measured by the reverse short-circuit current (RSCC) in urinary bladders from toads and turtles. Since these chemicals were equally effective under aerobic and anaerobic conditions, the inhibition appears to occur directly on UF0 rather than on mitochondria. In contrast, an inhibitor of F0-F1-ATPase, oligomycin, only inhibited aerobic RSCC and was ineffective on anaerobic RSCC. Thus, oligomycin appears to inhibit mitochondrial F0-F1 rather than any UF0+UF1. Another inhibitor of the F1-ATPase unit, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, was without effect on RSCC at a concentration (0.3 mM) that produced over 50% inhibition of short-circuit current. These results support the concept that acidifying urinary epithelia contain a plasma membrane proton channel, UF0, but that UF1, if it exists, is unique in that it is resistant to known inhibitors.