The human colon carcinoma cell line HT29 c1.19A was studied for organic anion transporter activity by determining intracellular fluo-3 and fura-red accumulation and by measuring fluo-3 efflux. Modulators of organic anion transport systems were used to identify the transporters that are involved in dye extrusion. Addition of probenecid to the dye-loading medium, containing 10 microM fluo-3/AM and fura-red/AM, resulted in a dose-dependent increase in fluo-3 and fura-red accumulation in the cells. The increase in fluo-3 accumulation in the cells in the presence of probenecid was explained by the inhibitory effect of this compound on fluo-3 efflux. Fluo-3 efflux from the cells was also inhibited by sulfinpyrazone, another inhibitor of organic anion transport. Substrates of renal probenecid-sensitive organic anion exchange mechanisms as well as modulators of multidrug resistance associated protein (MRP) activity did not influence fluo-3 extrusion rates. However, reducing intracellular ATP contents completely blocked fluo-3 extrusion. Moreover, MK571, an inhibitor of MRP, significantly stimulated dye accumulation, whereas inhibitors of the multidrug resistance gene (MDR1) product Pglycoprotein, cyclosporin A and verapamil, did not. As probenecid inhibits fluo-3 efflux across the apical membrane of cells grown on permeable supports, we conclude that a probenecid-sensitive organic anion transporter is present in the apical membrane of HT29 c1.19A cells. This organic anion transport system differs from MDRI and MRP2.