A double staining technique for simultaneously determinating cell surface phenotype and the degree of cell activation is described. As an activation marker, the tetrazolium dye MTT has been used. Cells were incubated for 30 min with MTT. Activated cells yielded a granular staining pattern. Upon termination of the reaction with sodium azide, a double-step immunofluorescence staining procedure using monoclonal antibodies specific for cell surface antigens was performed. The percentage of cells simultaneously displaying MTT formation and fluorescence was microscopically evaluated. Our results demonstrate that MTT staining is expressed concomitantly with the IL-2 receptor and the transferrin receptor. This method permits a simple characterization of activated T cell subsets and can be used clinically to analyse the T cell functions of patients being treated with immunosuppressive agents and patients with acquired immune deficiency syndrome.