Luminal isobutyrate, a relatively poor metabolized short-chain fatty acid (SCFA), induces HCO(3) secretion via a Cl-independent, DIDS-insensitive, carrier-mediated process as well as inhibiting both Cl-dependent and cAMP-induced HCO(3) secretion. The mechanism(s) responsible for these processes have not been well characterized. HCO(3) secretion was measured in isolated colonic mucosa mounted in Lucite chambers using pH stat technique and during microperfusion of isolated colonic crypts. (14)C-labeled butyrate, (14)C-labeled isobutyrate, and (36)Cl uptake were also determined by apical membrane vesicles (AMV) isolated from surface and/or crypt cells. Butyrate stimulation of Cl-independent, DIDS-insensitive 5-nitro-3-(3-phenylpropyl-amino)benzoic acid-insensitive HCO(3) secretion is greater than that by isobutyrate, suggesting that both SCFA transport and metabolism are critical for HCO(3) secretion. Both lumen and serosal 25 mM butyrate inhibit cAMP-induced HCO(3) secretion to a comparable degree (98 vs. 90%). In contrast, Cl-dependent HCO(3) secretion is downregulated by lumen 25 mM butyrate considerably more than by serosal butyrate (98 vs. 37%). Butyrate did not induce HCO(3) secretion in isolated microperfused crypts, whereas an outward-directed HCO(3) gradient-driven induced (14)C-butyrate uptake by surface but not crypt cell AMV. Both (36)Cl/HCO(3) exchange and potential-dependent (36)Cl movement in AMV were inhibited by 96-98% by 20 mM butyrate. We conclude that 1) SCFA-dependent HCO(3) secretion is the result of SCFA transport across the apical membrane via a SCFA/HCO(3) exchange more than intracellular SCFA metabolism; 2) SCFA-dependent HCO(3) secretion is most likely a result of an apical membrane SCFA/HCO(3) exchange in surface epithelial cells; 3) SCFA downregulates Cl-dependent and cAMP-induced HCO(3) secretion secondary to SCFA inhibition of apical membrane Cl/HCO(3) exchange and anion channel activity, respectively.