Transport of 35S-labeled sulfobromophthalein [35S]BSP was studied in short-term cultured rat hepatocytes incubated in bovine serum albumin. At 37 degrees C, initial uptake of [35S]BSP was 5-10-fold that at 4 degrees C, linear for at least 15 min, saturable, inhibited by bilirubin, and reduced by greater than 70% after ATP depletion or isosmotic substitution of sucrose for NaCl in medium. Replacement of Na+ by K+ or Li+ did not alter uptake, whereas replacement of Cl- by HCO-3 or gluconate- reduced uptake by approximately 40%. Substitution of Cl- by the more permeant NO-3 enhanced initial BSP uptake by 30%. Efflux of [35S]BSP from cells to media was inhibited by 40% after ATP depletion or sucrose substitution. To confirm these results in a more physiologic system, transport of [3H]bilirubin was studied in isolated livers perfused with control medium or medium in which Cl- was replaced by gluconate-. Perfusion data analyzed by the model of Goresky, revealed 40-50% reductions in influx and efflux with gluconate- substitution. These results are consistent with existence of a Cl-/organic anion-exchange mechanism similar to that described by others in renal tubules.