Biliary excretion of glutathione, free amino acids, and total amino acids (after acid hydrolysis) was measured in hepatic bile collected from guinea pigs, rabbits, and dogs anesthetized with pentobarbital sodium. In controls, the concentration of glutathione in bile was less than 20 microM in all three species. However, when hepatic gamma-glutamyltransferase activity was decreased by retrograde intrabiliary infusion of the irreversible inhibitor acivicin (AT-125; 20 mumol/kg), there was a marked increase in biliary glutathione excretion (in mumol glutathione equivalents.kg body wt-1.h-1) from 0.10 +/- 0.04 to 2.2 +/- 0.6 in guinea pigs, from 0.014 +/- 0.013 to 2.5 +/- 1.9 in rabbits, and from an undetectable level (less than 0.001) to 0.11 +/- 0.05 in dogs. Amino acid analysis of bile revealed that the concentration of glutathione's constituent amino acids (free glutamate, cystine, and glycine) in control bile samples from these three species were quite low and were not affected by AT-125. However, acid hydrolyzates of these same bile samples revealed an unusually high degree of amino acid conjugation. Glutamate (0.06-0.5 mM), cystine (0.2-1.1 mM), and glycine (1.7-2.8 mM) constituted the overwhelming majority of total amino acids in hydrolyzed bile from controls. After AT-125, concentrations of total glutamate and cystine were elevated in hydrolyzed bile, while concentrations of all other amino acids remained the same. Thus glutathione is avidly secreted into bile in the guinea pig, rabbit, and dog but is almost quantitatively broken down within the biliary tree. Subsequently, the glutamate and cysteine moieties derived from catabolism of glutathione must be partially reabsorbed either as peptides, conjugates, or free amino acids.(ABSTRACT TRUNCATED AT 250 WORDS)