Activation of murine spleen cells in vitro with soluble anti-CD3 monoclonal antibody and phorbol 12-myristate 13-acetate (PMA) induced an initial production of interleukin 2 (IL2), interferon-gamma (IFN-gamma), IL4 and IL5, followed by a refractory state during which the T cells did not produce lymphokines when stimulated with some common mitogens. The refractory state was long-lasting, depended on the presence of anti-CD3 and PMA but could be reverted by incubation in fresh medium. Pokeweed mitogen (PWM) differed from other mitogens tested, since stimulation by PWM and PMA induced lymphokine production and proliferation also in the refractory cells. Furthermore, PWM stimulation selectively induced IL4 and IFN-gamma production but not IL2 and IL5, as detected by intracellular cytokine-specific immunofluorescence and in situ hybridization for mRNA. The results indicate differential regulation of lymphokine production in primary lymphocytes.