Thrombin activates protease-activated receptors (PARs) by specific cleavage of their amino-terminal exodomains to unmask a tethered ligand that binds intramolecularly to the body of the receptor to effect transmembrane signaling. Peptides that mimic such ligands are valuable as agonists for probing PAR function, but the tethered ligand peptide for PAR4, GYPGKF, lacks potency and is of limited utility. In a structure-activity analysis of PAR4 peptides, AYPGKF was approximately 10-fold more potent than GYPGKF and, unlike GYPGKF, elicited PAR4-mediated responses comparable in magnitude to those elicited by thrombin. AYPGKF was relatively specific for PAR4 in part due to the tyrosine at position 2; substitution of phenylalanine or p-fluorophenylalanine at this position produced peptides that activated both PAR1 and PAR4. Because human platelets express both PAR1 and PAR4, it might be desirable to inhibit both receptors. Identifying a single agonist for both receptors raises the possibility that a single antagonist for both receptors might be developed. The AYPGKF peptide is a useful new tool for probing PAR4 function. For example, AYPGKF activated and desensitized PAR4 in platelets and, like thrombin, triggered phosphoinositide hydrolysis but not inhibition of adenylyl cyclase in PAR4-expressing cells. The latter shows that, unlike PAR1, PAR4 couples to G(q) and not G(i).