Prodigiosin, prodigiosin 25-C, and metacycloprodigiosin all strongly inhibited the acidification activity of (H + +K + )-ATPase on membrane vesicles from hog gastric mucosa (ic 5 0 = 32 to 103 pmol/mg protein). But, the prodigiosins, unlike omeprazole, showed little inhibitory effect on K + -dependent ATPase (K + -ATPase) activity, although at higher concentrations they inhibited K + -ATPase activity with an ic 5 0 of 1.5 to 3.0 μM. Furthermore, the inhibitory effect of the prodigiosins was rapid and completely reversible unlike that of omeprazole, and the mode of inhibition was non-competitive with respect to ATP. Hog gastric (H + +K + )-ATPase itself showed an absolute requirement of halide (effectively, chloride) for acidification activity. Prodigiosins also showed a chloride requirement for inhibition of vesicular acidification, and quickly reversed the acidification of vesicular pH to neutrality even in the presence of N,N'-dicyclohexylcarbodiimide (DCCD), showing their ionophoric nature of acidification inhibitory activity. In fact, tributyltin chloride (TBT, an OH - /Cl - exchange ionophore) also inhibited vesicular acidification, but it inhibited K + -ATPase activity too. Finally, the prodigiosins inhibited the acid secretion from parietal cells isolated from rabbit gastric mucosa. These results suggest that prodigiosins are potent reversible uncouplers of (H + +K + )-ATPase that inhibit gastric acid secretion.