The potential role of phospholipid metabolism in restricting lymphocyte proliferation under conditions of oxidative stress was investigated using [1-14C]-arachidonic acid (14C-AA) and 32P-orthophosphoric acid. Human peripheral blood lymphocytes (PBL) and PBL depleted of glutathione with L-buthionine-S,R-sulfoximine (BSO-PBL) were compared. The relative uniformity of glutathione depletion in the PBL population was assessed by flow cytometry. BSO-PBL were 40 to 90% depleted of glutathione 1 to 3 days after activation, respectively, and the BSO-PBL had unimpaired early activation events based on 32P-phosphatidylinositol levels. However, unlike stimulated PBL, which showed a progressive decrease in radioactivity incorporated into phosphatidylcholine and a corresponding increase into phosphatidylethanolamine, no significant differences occurred with BSO-PBL. Prelabeled BSO-PBL showed considerably more 14C radioactivity in the supernatant following 72-120 h stimulation with anti-CD3 than control PBL, which was mostly in the form of unmetabolized 14C-AA. Higher levels of leukotriene B4, 5-hydroxyeicosatetraenoate and 12-hydroxy-5,8,10-heptadecatrienoate also were observed with L-buthionine-S,R-sulfoximine treatment, which could explain the impaired proliferation obtained with a depletion of cellular glutathione. Both lysophosphatidylcholine and liberated free 14C-AA increased with L-buthionine-S,R-sulfoximine treatment following 72 h stimulation, suggesting functional impairment in the reacylating enzymes. The increased release of 14C-AA by BSO-PBL also may contribute to the imparied proliferation that occurs with loss of glutathione.