Sulfated and non sulfated lithocholic acid were analyzed in serum, bile and urine of bile-fistula Sprague-Dawley female rats by gas-liquid chromatography and thin-layer chromatography. In rats (group A) bile was collected during a 3 h saline infusion followed by a 3 h intravenous infusion of taurolithocholate; animals of group B and C were infused with both taurolithocholate and rifamycin-SV, but in the animals of group C the renal pedicles were ligated. Serum bile acids showed a significant increase only in animals of group C, during the infusion of rifamycin-SV. In bile of animals of group A sulfated lithocholic acid represented 18% of total, but was not found in bile of animals of group B and C, while a significant increase of sulfated lithocholic acid was evident in urine of animals of group B. The results indicate that rifamycin-SV probably interferes with the hepatic secretion of sulfated lithocholic acid in the rat.