The Ras-like guanine-nucleotide-binding protein Rap1 controls integrin alpha(IIb)beta3 activity and platelet aggregation. Recently, we have found that Rap1 activation can be blocked by the nitric oxide/cyclic guanosine monophosphate (NO/cGMP) signaling pathway by type 1 cGMP-dependent protein kinase (cGKI). In search of possible targets of NO/cGMP/cGKI, we studied the expression of Rap1-specific GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs) in platelets. We could detect mRNAs for a new protein most closely related to Rap1GAP and for postsynaptic density-95 discs-large and zona occludens protein 1 (PDZ)-GEF1 and CalDAG-GEFs I and III. Using 5'-rapid amplification of cDNA ends (RACE), we isolated the complete cDNA of the new GAP encoding a 715-amino acid protein, which we have termed Rap1GAP2. Rap1GAP2 is expressed in at least 3 splice variants, 2 of which are detectable in platelets. Endogenous Rap1GAP2 protein partially colocalizes with Rap1 in human platelets. In transfected cells, we show that Rap1GAP2 exhibits strong GTPase-stimulating activity toward Rap1. Rap1GAP2 is highly phosphorylated, and we have identified cGKI as a Rap1GAP2 kinase. cGKI phosphorylates Rap1GAP2 exclusively on serine 7, a residue present only in the platelet splice variants of Rap1GAP2. Phosphorylation of Rap1GAP2 by cGKI might mediate inhibitory effects of NO/cGMP on Rap1. Rap1GAP2 is the first GTPase-activating protein of Rap1 found in platelets and is likely to have an important regulatory role in platelet aggregation.