Bilirubin and phthalein dyes are taken up by the liver via a carrier-mediated mechanism operated at least in part by bilitranslocase (BTL). Because they also undergo renal transport, the presence and function of BTL was investigated in rat renal tubular plasma membrane vesicles. Transport of sulfobromophthalein (BSP) was enriched in basolateral domain of plasma membrane and followed the distribution pattern of Na(+)-K(+)-ATPase but not of gamma-glutamyltransferase. BSP uptake was inhibited by addition of monospecific antibodies raised against hepatic BTL. As in liver vesicles, BSP transport was electrogenic, being greatly accelerated by addition of valinomycin in presence of an inwardly directed K+ gradient. Apparent Km of BSP transport was 17 +/- 2 microM (n = 3 expts), one order of magnitude higher than that measured in liver; however, Vmax was similar to that described in liver vesicles (429 +/- 18 nmol BSP.mg protein-1.min-1, n = 3 expts). Competitive inhibition was observed with both unconjugated bilirubin (Ki, 2.9 +/- 0.2 microM) and rifamycin SV (Ki, 76 +/- 10 microM), known competitors for hepatic BTL-mediated transport of BSP. Immunoblotting studies with anti-BTL monospecific antibodies revealed presence of a single positive band only in basolateral-enriched membrane fraction; its apparent molecular mass was 37 kDa, virtually identical to that of hepatic protein. Immunohistochemistry confined presence of BTL to renal proximal tubules (RPT) We conclude that BTL is present in basolateral plasma membrane of RPT cells. Lower affinity of renal, compared with hepatic protein, for substrates might explain the marginal role of kidney in plasma clearance of bilirubin and cholephilic dyes.