Alveolar epithelial lining fluid glutathione (GSH) is markedly decreased in patients with idiopathic pulmonary fibrosis (IPF). Because patients with IPF have exaggerated numbers of fibroblasts in their lower respiratory tract, we hypothesized that GSH can suppress lung fibroblast proliferation. To verify this hypothesis, we examined the ability of GSH to suppress human lung fibroblast (ATCC; HFL-1) proliferation in vitro in the presence of either IPF bronchoalveolar lavage fluid (BAL) or calf serum (CS). Both CS at a concentration of 10% and IPF BAL markedly increased fibroblast proliferation when compared to cells grown without CS or IPF BAL (10% CS = 93 ± 4%, P < 0.001; IPF BAL = 47 ± 4%, P < 0.001). In the presence of physiologic concentrations of GSH (0 to 500 µM), both CS- and IPF BAL-mediated fibroblast proliferation were markedly reduced, with 500 µM GSH inducing complete inhibition. Interestingly, glutathione disulfide (GSSH) and S-methylglutathione did not suppress proliferation, whereas various sulfhydryl-containing molecules (cysteine, N-acetylcysteine, 2-mercaptoethanol, and low concentrations of dithiothreitol) induced an inhibition of fibroblast proliferation similar to that observed with GSH. Most of the suppressive effect of GSH was mediated at the cell level since incubation of fibroblasts with 500 µM GSH for 1 h completely blocked the ability of the cells to subsequently proliferate in the presence of untreated 10% CS. Treatment of CS with 500 µM GSH for 1 h followed by removal of GSH by molecular sieve chromatography had no detectable effect on fibroblast proliferation. Catalase inhibited the GSH-mediated suppression of fibroblast proliferation, suggesting that oxidation of the sulfhydryl group may produce H₂O₂-related oxidants, which could inhibit fibroblast proliferation. We conclude that (1) GSH in the extracellular milieu can suppress lung fibroblast proliferation in response to mitogens and (2) suppression is related to the presence of the sulfhydryl group of GSH. It is possible that a lower-respiratory-tract GSH deficiency as observed in IPF may create an environment favoring excessive fibroblast proliferation.