The effect on HCO3- absorption of the substituted benzimidazole omeprazole, an inhibitor of active H+-K+ exchange, was examined in in vitro Amphiuma jejunum. HCO-3 absorption was measured using titration in short-circuited intestinal segments bathed in a Cl--free (SO2-(4)-based) medium (pH 7.40). At 1 mM omeprazole lowered the short-circuit current (Isc) and the absorptive flux of HCO3- from mucosa to serosa by about 40% over 1 h. When the serosal medium was maintained at pH 5.0, additions of omeprazole beginning at 0.1 mM caused proportional reductions of the Isc. Omeprazole also reduced intracellular potassium activity (aiK) from 74 to 59 mM and lowered the luminal membrane potential (psi m) slightly over 30 min when this was measured with double-barreled, K+-sensitive microelectrodes. After 30 min aiK and psi m tended to spontaneously revert toward control values. Galactose added to the mucosal medium (to 20 mM) stimulated the Isc equally in omeprazole-treated tissues and paired untreated control tissues. These results support the view that a fraction of the absorptive HCO3- flux (so-called) is driven by an active luminal exchange of H+ for K+.