Current studies were undertaken to characterize the mechanism of short-chain fatty acid (SCFA) transport in isolated human proximal colonic basolateral membrane vesicles (BLMV) utilizing a rapid-filtration n-[¹⁴C]butyrate uptake technique. Human colonic tissues were obtained from mucosal scrapings from organ donor proximal colons. Our results, consistent with the existence of a HCO/SCFA exchanger in these membranes, are summarized as follows: 1) n-[¹⁴C]butyrate influx was significantly stimulated into the vesicles in the presence of an outwardly directed HCO and an inwardly directed pH gradient; 2) n-[¹⁴C]butyrate uptake was markedly inhibited (~40%) by anion exchange inhibitor niflumic acid (1 mM), but SITS and DIDS (5 mM) had no effect; 3) structural analogs e.g., acetate and propionate, significantly inhibited uptake of HCO and pH-gradient-driven n-[¹⁴C]butyrate; 4) n-[¹⁴C]butyrate uptake was saturable with a K_m for butyrate of 17.5 ± 4.5 mM and a V_max of 20.9 ± 1.2 nmol · mg protein¹ · 5 s¹; 5) n-[¹⁴C]butyrate influx into the vesicles demonstrated a transstimulation phenomenon; and 6) intravesicular or extravesicular Cl did not alter the anion-stimulated n-[¹⁴C]butyrate uptake. Our results indicate the presence of a carrier-mediated HCO/SCFA exchanger on the human colonic basolateral membrane, which appears to be distinct from the previously described anion exchangers in the membranes of colonic epithelia.