Mitogen-induced alterations in protein phosphorylation were studied in purified human peripheral blood lymphocytes. Cells were prelabeled with [32P]-orthophosphate, treated with mitogens for varying times, and disrupted by sonication in a medium that inhibited further phosphorylation or dephosphorylation. Sonicates were separated into soluble and particulate fractions by centrifugation at 16,000 x G. [32P]-content of phospho-proteins in both the particulate and soluble fractions was analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. Treatment of lymphocytes with concanavalin A, phytohemagglutinin, rabbit anti-human β2-microglobulin, and the divalent cation ionophore A23187, all mitogenic agents, resulted in a rapid, dose dependent increase in protein phosphorylation seen over the mitogenic range. Increased phosphorylation was detected only in the soluble fraction of sonicates and consisted of modest increases in [32P]-content of a large number of proteins ranging in m.w. from 30,000 to 200,000. In contrast to these changes, phosphorylation of one discrete protein with m.w. of approximately 65,000 was markedly stimulated in cells treated with mitogenic agents. Mitogens were not found to stimulate the phosphorylation of any major particulate proteins, including the β2-microglobulin-associated phosphoproteins. Increases in phosphorylation of soluble proteins produced by concanavalin A, anti-β2-microglobulin, and phytohemagglutinin were maximal at 10 min, whereas A23187-induced increases were maximal within 3 to 5 min. Treatment of lymphocytes with prostaglandin E1 immediately before treatment with concanavalin A resulted in inhibition of the concanavalin A-induced phosphorylation response. Neither isoproterenol nor N6-monobutyryl cyclic AMP demonstrated similar inhibitory activity. Neither cyclic GMP nor sodium nitroprusside altered phosphorylation in the presence or absence of concanavalin A. The proteins whose phosphorylation is altered in cells treated with mitogenic agents may occupy an important role in the initiation of the proliferative response.