Histamine, beta adrenergic amines, and prostaglandins inhibited hemolytic plaque formation by splenic leukocytes from immunized mice. The same agents had previously been shown to prevent both the IgE mediated release of histamine from human basophils and the immunologically specific cytolytic activity of murine lymphocytes, through stimulation of the production of cyclic AMP in leukocytes. The authors therefore tested the hypothesis that cyclic AMP might mediate an inhibitory effect of these drugs by comparing the ability of these agents to inhibit plaque formation with their effects on cyclic AMP accumulation in leukocytes. In splenic cells from three mouse 3α,17β dose dependent effects of these agents on cyclic AMP correlated with their inhibition of plaque formation. Beta but not alpha adrenergic agonists were effective in both systems, and the effects of isoproterenol were inhibited by propranolol. Histamine was approximately equipotent with isoproterenol in both systems. Two prostaglandins (E1 and E2) were effective in both systems, but prostaglandin F(2α) was not. Dibutyril cyclic AMP, a lipid soluble analog of the endogenous nucleotide, inhibited plaque formation by cells of all three strains. Theophylline, an inhibitor of cyclic AMP degradation, inhibited plaque formation slightly, but potentiated the effects of histamine, isoproterenol, and the prostaglandins on both cyclic AMP accumulation and plaque formation. Finally, cholera enterotoxin, a potent activator of adenyl cyclase, produced a delayed inhibition of plaque formation and a parallel increase in leukocyte cyclic AMP content; both effects of the toxin were blocked by canine antitoxin. These results suggest that leukocyte cyclic AMP may act as a 'second messenger' to suppress plaque formation in vitro. The inhibitory effects of hormones and cyclic AMP on plaque formation were strikingly similar to their effects on in vitro models of immediate and cell mediated hypersensitivity. The physiological significance of these findings is not yet known.