Hepatic stellate cells are resistant to the fibrogenic effects of lipid hydroperoxides in primary culture. Recent studies from our laboratory suggest that antioxidants, particularly glutathione, play a role in this resistance. We have observed that glutathione accumulates rapidly in stellate cells during primary culture; in the current study, we investigated whether glutathione modulates stellate cell collagen synthesis. Stellate cells from normal rat liver were plated in primary culture and maintained for 7 days. From day 4 through day 7, the cells were treated with L-buthionine sulfoximine (BSO) to deplete glutathione stores. BSO profoundly diminished stellate cell glutathione but had no effect on morphology, viability, or basal levels of collagen synthesis and gene expression. When cultured stellate cells were incubated with the putative fibrogenic mediator 4-hydroxynonenal or iron/ascorbate, little or no increase in collagen synthesis occurred regardless of glutathione content. In contrast, iron/ascorbate induced collagen synthesis by cultured fibroblasts. The data indicate that stellate cells strongly resist oxidant- and lipid peroxide-induced collagen synthesis in primary culture. They demonstrate that the mechanism of this resistance does not involve glutathione.