Platelets were used to study the activation of Rho and Rac through G-protein-coupled receptors and its regulation by cyclic nucleotides. The thromboxane A2 (TXA2) mimetic U46619 rapidly activated both small GTPases independently of integrin aIIbb3 activation. U46619 which leads to the activation of G12/G13 and Gq did not induce Rac activation in Gaq-deficient platelets but was able to activate Rho, to stimulate actin polymerization and phosphatidylinositol-4,5-bisphosphate formation and to induce shape change. Rac activation by U46619 in wild-type platelets could be blocked by chelation of intracellular Ca2+ and was partially sensitive to apyrase and AR-C69931MX, an antagonist of the G i-coupled ADP receptor. Cyclic AMP which completely blocks platelet function inhibited the U46619-induced activation of Gq and G12/G13 as well as of Rac and Rho. In contrast, cGMP which has no effect on platelet shape change blocked only activation of Gq and Rac. These data demonstrate that Rho and Rac are differentially regulated through heterotrimeric G-proteins. The G12/G13-mediated Rho activation is involved in the shape change response, while Rac is activated through Gq and is not required for shape change. Cyclic AMP and cGMP differentially interfere with U46619-induced Rho and Rac activation at least in part by selective effects on the regulation of individual G-proteins through the TXA2 receptor.