Cellular thiols have long been known to play a role in cell activation and proliferation; however, the differential expression of surface thiols on the lymphoid subsets had not been described. Neither was it known whether alteration of surface thiols occurs after exposure to mitogens or infectious agents. Herein, an impermeant thiol-specific fluorescent probe was employed for flow cytometric analysis of surface thiols. Quantification of surface thiols on resting lymphocytes revealed that some subsets expressed different concentrations of surface thiols (CD19+ > CD8+ > CD4+). Furthermore, surface thiols increased on all subsets by 8 h after mitogenic activation. This increase was blocked by cycloheximide or monensin but not by actinomycin D or inhibition of glutathione synthesis by buthionine sulfoximine. In addition, bacitracin, an inhibitor of protein disulfide isomerase, inhibited the increase in surface thiols and DNA synthesis. Lymphocytes from HIV-infected individuals displayed increased surface thiols on CD19+ and CD4+ cells but not CD8+ cells. Although cellular thiols in general have been believed to play a role in protection against oxidants, signaling associated with cell growth, and apoptosis, there is now evidence that changes in exofacial thiols appear to be involved in some of these critical cell reactivities. Thus, quantitative and possibly qualitative differences in surface thiols correlate with membrane differences between lymphoid subsets and with their differential sensitivities to oxidative stress, which suggests that the mechanisms by which surface thiols are maintained and modified after activation are important cellular functions that need to be further evaluated.