Thrombin receptor activation, by thrombin or SFLLR-containing peptides, stimulates GTPase activity in platelet and CHRF-288 membranes. Polyclonal antibodies to peptides derived from the thrombin receptor (anti-TR52-69 and anti-TR36-49), which block many of thrombin's actions on platelets and endothelial cells, also block thrombin activation of membrane GTPase (as does thrombin active site and anion-binding exosite inhibitors). Most of the receptor-activated GTPase, stimulated by both thrombin and SFLLRNP in platelet membranes, was inhibited by prior treatment with pertussis toxin or N-ethylmaleimide, suggesting that under these conditions much of the thrombin receptor-stimulated GTPase in platelet membranes is a member of the pertussis toxin-sensitive G alpha i family. In platelet membrane preparations, the peptide agonists stimulated approximately twice as much GTPase activity as stimulated by alpha-thrombin. In contrast, the membranes prepared from CHRF-288 cells showed similar maximal SFLLRNP- and alpha-thrombin-stimulated GTPase activity. Stimulation of the platelet membrane GTPase by a variety of different peptide agonists correlated with their ability to stimulate platelet aggregation. Several peptide-based agonists were more potent than the wild-type sequence. The most potent was Ser-(p-fluoro-Phe)-(2-Napthyl-Ala)-Leu-Arg-NH2, which stimulated platelet aggregation (EC50 = 80 nM) and GTPase activity (EC50 = 110 nM). The peptide YFLLRN stimulated GTPase activity but only to approximately 40% of the activity observed with optimal concentrations of other receptor agonists. YFLLRN also limited the stimulation observed with SFLLRNP in a competitive fashion, indicating that YFLLRN is a competitive partial agonist at the thrombin receptor. These studies show that the tethered-ligand receptor mediates the GTPase activation by thrombin in platelet and CHRF-288 cell membranes, and this provides a specific, reliable, and convenient cell-free assay system with which one can evaluate agonists and partial agonists.