Human serum albumin (HSA) is a cystine-rich serum protein taken up by many cells through receptor-mediated and fluid-phase endocytosis. We hypothesized that HSA may play a role in modulating cellular antioxidant redox signaling. Lung epithelial cells (A549), fibroblasts (HFL1), and blood lymphocytes had increased glutathione (GSH) levels after 8 h incubation with HSA. Similar GSH increases were observed with either plasma-derived or recombinant HSA. Serum depleted of HSA had no effect on cellular GSH. The GSH increase was also observed in normal murine lungs upon in vivo airway instillation of HSA. GSH enhancement was not related to the redox state of the free cysteine residue (Cys-34) on HSA, however, reduction of disulfide bonds in HSA inhibited the increase in cellular GSH. In addition, the albumin-mediated increase in GSH was inhibited by the vacuolar (H(+))-ATPase inhibitors, bafilomycin A(1) and concanamycin, as well as by the membrane pH-disrupting ionophore monensin, but not by 20 mM NH(4)Cl. The degree to which albumin increased GSH levels was sufficient to protect cells against H(2)O(2)-mediated cytotoxicity and to decrease TNF-alpha-mediated NF-kappaB activation. We conclude that albumin specifically modulates cellular GSH levels, an effect sufficient to protect cells against oxidant injury and regulate NF-kappaB activation.