Biosynthetic labeling with 35S-methionine and 35S-cysteine of isolated T4+ cells from Con A-activated T cells demonstrated that the T8 antigen was synthesized by activated T4+ cells. Two-color fluorescence analysis of the activated T cell population from which the T4+ fraction was obtained showed that both T4+T8- and T4+T8+ cells were present. The T8 antigen that was immunoprecipitated by monoclonal anti-T8 from activated T4+ cells migrated with an electrophoretic mobility corresponding to an m.w. of approximately 33,000, a previously reported m.w. value for T8 antigen. Con A activation of highly purified peripheral T4+T8- and T8+T4- subsets indicated that both T4+T8- and T8+T4- cells can give rise to T4+T8+ cells. However, substantial T4, T8 coexpression by T4+T8- cells required a signal from T8+T4- cells which could be supplied by incubating T4+T8- cells with irradiated T8+ cells or the supernatant from Con A-activated T8+T4- cells. The generation of T4+T8+ cells from a subset of T4+T8- T cells may be an important mechanism in immune activation and/or the further differentiation of peripheral T4+ cells.