1. The first step in the bioactivation of the nephrotoxin hexachlorobutadiene is the biosynthesis of 1-(glutathion-S-yl)-1,2,3,4,4-pentachloro-1,3-butadiene (GPCB). GPCB formed in the liver is secreted into bile, may be reabsorbed in the gut, intact or after hydrolysis to 1-(cystein-S-yl)-1,2,3,4,4-pentachloro-1,3-butadiene (CPCB), and undergo enterohepatic circulation or translocation to the kidney. Hepatic uptake and metabolism of GPCB and CPCB may thus influence the disposition of these S-conjugates. We therefore studied the metabolism and uptake of CPCB and GPCB in the isolated perfused rat liver. 2. Dose-dependent uptake of GPCB and CPCB from the perfusion medium by isolated perfused liver was demonstrated; CPCB is cleared from the perfusion medium to a much higher extent than GPCB. 3. GPCB and CPCB are intensively biotransformed to biliary metabolites. These metabolites were identified by thermospray mass spectrometry as products of the conjugation reaction of GPCB and CPCB with glutathione and subsequent hydrolysis of the glutathione moieties. 4. Hepatic biosynthesis of 1-(N-acetyl-L-cystein-S-yl)-1,2,3,4,4-pentachloro- 1,3-butadiene from CPCB was only a very minor pathway in GPCB and CPCB metabolism in liver. 5. The results indicate that hepatic biosynthesis of mercapturic acids may not contribute to the disposition of S-conjugates formed from hexachlorobutadiene in vivo and that GPCB may be, at least in part, delivered intact to the kidney.