In blood platelets the small GTPase Rap1b is activated by cytosolic Ca2+ and promotes integrin αIIbβ3 inside-out activation and platelet aggregation. cAMP is the major inhibitor of platelet function and antagonizes Rap1b stimulation through a mechanism that remains unclear. In the present study we demonstrate that the Ca2+-dependent exchange factor for Rap1b, CalDAG-GEFI (calcium and diacylglycerol-regulated guanine-nucleotide-exchange factor I), is a novel substrate for the cAMP-activated PKA (protein kinase A). CalDAG-GEFI phosphorylation occurred in intact platelets treated with the cAMP-increasing agent forskolin and was inhibited by the PKA inhibitor H89. Purified recombinant CalDAG-GEFI was also phosphorylated in vitro by the PKA catalytic subunit. By screening a panel of specific serine to alanine residue mutants, we identified Ser116 and Ser586 as PKA phosphorylation sites in CalDAG-GEFI. In transfected HEK (human embryonic kidney)-293 cells, as well as in platelets, forskolin-induced phosphorylation of CalDAG-GEFI prevented the activation of Rap1b induced by the Ca2+ ionophore A23187. In platelets this effect was associated with the inhibition of aggregation. Moreover, cAMP-mediated inhibition of Rap1b was lost in HEK-293 cells transfected with a double mutant of CalDAG-GEFI unable to be phosphorylated by PKA. The results of the present study demonstrate that phosphorylation of CalDAG-GEFI by PKA affects its activity and represents a novel mechanism for cAMP-mediated inhibition of Rap1b in platelets.