Previous studies in cultured rat hepatocytes revealed that initial uptake of sulfobromophthalein (BSP) was markedly reduced upon removal of Cl− from the medium. In the present study, unidirectional Cl− gradients were established in short-term cultured rat hepatocytes and their effect on BSP uptake was determined. These investigations revealed that BSP uptake requires external Cl− and is not stimulated by unidirectional Cl− gradients, suggesting that BSP transport is not coupled to Cl− transport. In contrast, BSP transport is stimulated by an inside-to-outside OH− gradient, consistent with OH− exchange or H+ cotransport. As the presence of Cl− is essential for but not directly coupled to BSP transport, binding of 35S-BSP to hepatocytes was determined at 4° C. This revealed an ∼ 10-fold higher affinity of cells for BSP in the presence as compared to the absence of Cl− (Ka + 3.2 ± 0.8 vs. 0.42 ± 0.09 μM−1; P < 0.02). Affinity of BSP for albumin was Cl−-independent, and was ∼ 10% of its affinity for cells in the presence of Cl−. These results indicate that extracellular Cl− modulates the affinity of BSP for its hepatocyte transporter.