In vitro T cell activation requires both antigen presentation and a second stimulus provided by the lymphocyte costimulator. Neither alone is sufficient to induce specific T cell activation. The S+ phenotype of stimulating cells is dependent on the metabolic activity of these cells. This finding is consistent with the notion that production and/or release of the costimulator is a function of metabolically active cells. The costimulator acts at an early stage of the interaction between lymphocyte and antigen, and the costimulator, or a separate maintenance factor, is required throughout the culture period for the expression of full cytotoxic activity. The lymphocyte costimulator is not strain specific but is phylogenetically specific. The activation of cytotoxic T cells by S+ cells is also phylogenetically specific, and this specificity of cellular activation can be accounted for by the species specificity of the lymphocyte costimulator.