This paper examines the hypothesis that reactive oxygen species (ROS) play an important role as second messengers in T cell activation. Activation of T cells with phorbol ester in combination with either calcium ionophore, or anti-CD3 antibody results in a large rapid flux of ROS activity. In contrast, co-stimulation with CD28 does not enhance ROS activity. The ROS signal was sensitive to ascorbic acid, desferrioxamine and dimethyl sulfoxide, suggesting that the major active species being generated was the hydroxyl radical, probably by iron-catalyzed decomposition of hydrogen peroxide. The generation of ROS in T cells was regulated by an accessory population within the peripheral blood. An anti-CD2 antibody induced a strong ROS flux, suggesting that the CD2/LFA-3 interaction may be important in this regulation. T cell activation was inhibited by the same panel of anti-oxidants as ROS generation, but much higher concentrations were required for inhibition of proliferation and IL-2 release than those required to block ROS generation. These data imply that ROS are not obligate second messengers for initiation of T cell activation. The results are compatible, however, with a role for activation-dependent T cell ROS generation in modulating the overall T cell response via autocrine and paracrine signalling pathways.