Reduced and oxidized glutathione contents of adult rat hepatocytes in pure culture and in co-culture with rat epithelial cells were measured under various medium conditions. To the standard medium fetal calf serum, nicotinamide, H2Se03, dimethylsulphoxide or no supplements were added. For freshly isolated hepatocytes, intracellular contents of 24 Β± 7 nmol reduced and O. 7 +_ 0.2 nmol oxidized glutathione/ mg cellular protein were obtained, respectively. In pure culture as well as in co-culture and regardless of the medium conditions involved, the protein content stays constant during the culture time with the exception of a decrease in protein content after 6 days of pure culture, caused by deterioration and loss of the hepatocytes. In both culture systems, an initial increase in intracellular reduced glutathione levels was observed, followed by a decrease and a quick normalisation in co-culture. On the contrary, in pure culture, the decrease was slower, but not transient and a stabilized situation was never reached. The various supplementations of the culture media had no significant effect on the intracellular reduced glutathione contents of both culture systems. As far as the intra-and the extracellular oxidized glutathione contents and the extracellular reduced form are concerned, these were only present in small amounts.