Adenosine 3':5'-cyclic monophosphate (cyclic AMP) has been shown to have an antimitotic role in various cell types, and it has been hypothesized that a decrease of cyclic AMP concentration in the cell initiates or permits cell division. This hypothesis has been evaluated with respect to clonal proliferation of lymphocytes. Two potent mitogenic agents, phytohemagglutinin and concanavalin A, which induce thymic-dependent lymphocytes to undergo clonal proliferation, were examined for their ability to initiate proliferation and to alter the concentrations of cyclic AMP and guanosine 3':5'-cyclic monophosphate (cyclic GMP) in purified human peripheral blood-lymphocytes. Optimal mitogenic concentrations of phytohemagglutinin and concanavalin A produced 10- to 50-fold increases in the concentration of lymphocyte cyclic GMP within the first 20 min of exposure to the mitogens. No changes were seen in the concentration of cyclic AMP after stimulation with either mitogen in purified form. Increases of less than 2-fold in the concentration of lymphocyte cyclic AMP observed with a less purified preparation of phytohemagglutinin could be attributed to the agglutinating rather than the mitogenic properties of the mitogen. A revised hypothesis is presented in which a temporally discrete rise in lymphocyte cyclic GMP concentration is viewed as an active signal to induce proliferation, while the elevation of cyclic AMP concentration in these cells is viewed as a regulatory influence that limits or inhibits mitogenic action.