Ascorbic acid and serotonin increased the 3′,5′-guanosine monophosphate (cGMP) content and enhanced the chemotactic responsiveness of mononuclear cells from human peripheral blood. The ionophore A23187, PGE1, and polystyrene beads increased the 3′,5′-adenosine monophosphate (cAMP) content of mononuclear cells and had no effect on basal cGMP content. A23187, PGE1, and beads caused significant increases in cAMP content in preparations of adherent cells (chiefly monocytes); PGE1 and beads also increased cAMP content in the nonadherent cells (chiefly lymphocytes). A23187 and PGE1 each inhibited mononuclear cell locomotion. The chemically unrelated agents, A23187, beads, and PGE1, that raised cAMP of monocytes inhibited the accumulation of cGMP in response to serotonin and asorbic acid. PGE1 at a concentration that caused a 349% increase in cAMP inhibited the effect of serotonin on cGMP accumulation and the amplification of chemotaxis by serotonin only 37% and 40%, respectively. In contrast A23187, which increased monocyte cAMP by only 71%, inhibited the effect of serotonin on cGMP accumulation and locomotion by 85 and 79%, respectively. Although there is no clear quantitative relationship between the increase in cAMP content and the degree of inhibition of cGMP accumulation, it is possible that the elevation of cAMP in monocytes interferes with their capacity to accumulate cGMP in response to serotonin and ascorbic acid, perhaps by increasing cGMP degradation and/or inhibiting synthesis. In any case cyclic nucleotide modulation of monocyte locomotion appears to be more closely related to intracellular cGMP content than to cAMP content.