Previous studies from our laboratory have shown that thrombin mitogenesis requires both high-affinity receptor occupancy and enzymic activity. Combined addition of DIP-inactivated-thrombin, which retains the ability to bind to thrombin receptors, and enzymically active gamma-thrombin generates a complete set of signals sufficient to initiate cell proliferation. Several possible signals, including stimulation of ion fluxes and phosphoinositide turnover, appear to be stimulated by thrombin's enzymic activity, but not by receptor occupancy. We now report that alpha-thrombin and DIP-thrombin stimulate an early, transient increase of 60 to 200% in intracellular levels of cAMP. This stimulation occurs at low mitogenic concentrations of alpha-thrombin where less than half the receptors are occupied. Enzymically active gamma-thrombin, which stimulates other types of signals, has no stimulatory effects on cAMP. Thus, this effect appears to be generated by high-affinity interaction of thrombin with its cell-surface receptors. Artificially increasing cAMP levels within these cells, however, cannot replace the requirement for thrombin-receptor occupancy in completing the mitogenic stimulation. Therefore, thrombin-receptor occupancy may generate additional, as yet unidentified, required signals.