The effect of galactosamine on the resynthesis of glutathione in rat primary hepatocyte cultures was investigated. Cultured rat hepatocytes were treated with galactosamine (4 mM) 1.5 hr prior to concurrent with, or 1.5 hr after cell attachment; total cellular glutathione was then measured over time. Addition of galactosamine at any of these times suppressed methionine-enhanced glutathione resynthesis in the cultures after a lag period of about 120 min. The lag period was not due to slow uptake of galactosamine by the cultured cells, since cellular UTP levels fell to less than 10% of controls within 60 min, a time frame comparable to that observed in vivo. Neither was the lag period a result of interference with cellular uptake of methionine or with conversion of methionine to cysteine, since the phenomenon was observed regardless of whether methionine or cysteine was used to promote glutathione resynthesis. Addition of uridine, which protects against galactosamine hepatotoxicity in vivo by replenishing hepatic UTP levels, did not prevent the suppression of glutathione resynthesis. The data indicate that (a) galactosamine inhibits the time-dependent resynthesis of glutathione in primary hepatocyte cultures, (b) a lag period exists for this response, and (c) this effect is not directly related to depletion of cellular UTP stores.