Using the isolated perfused rat liver technique we examined whether the accumulation and sinusoidal efflux processes of the organic anion dibromosulfophthalein (DBSP) are dependent on the intracellular ATP content, chloride concentration in the perfusion medium as well as temperature of the medium and whether they are mediated by the same or by separate carrier mechanisms. The net sinusoidal efflux rate, being the resultant of sinusoidal efflux and re-uptake, was decreased more than 50% after lowering the medium temperature from 37 to 26 degrees indicating that the efflux process is carrier-mediated. The uptake rate was decreased only 18% after lowering the medium temperature to 26 degrees. Lowering of the hepatic ATP content for more than 80% clearly decreased the DBSP uptake rate but not the sinusoidal efflux rate. These observations indicate that these opposing transport steps probably are mediated by two separate carriers. Additional evidence for this hypothesis originated from the observation that sinusoidal efflux of DBSP was decreased about 30% whereas hepatic uptake of the substrate remained unaltered after replacing chloride in the perfusion medium with gluconate. In summary, we conclude that uptake and sinusoidal efflux of DBSP are mediated by two separate carrier systems that are influenced differently by ATP depletion, temperature lowering and presence of Cl-gradients.