We studied the actions of the human and murine proteinase-activated receptor 4 (PAR4) derived receptor-activating peptides (APs), GYPGQV-NH2(GQV-NH2) and GYPGKF-NH2(GKF-NH2), (i) to activate-desensitize either PAR1or PAR2in cultured cell systems (calcium signalling in PAR1/PAR2-bearing human HEK cells and in rat KNRK cells expressing either rat or human PAR2) and (ii) to affect contractility in rat aorta (RA) and rat gastric longitudinal muscle (LM) preparations in vitro. We found that neither PAR1nor PAR2was affected by concentrations of the PAR4-APs (800 µM) that caused both an endothelium-dependent nitric oxide mediated relaxation of preconstricted RA tissue and a contractile response in the LM preparation. The potencies (EC50values 300 to 400 µM) of GQV-NH2and GKF-NH2for causing a relaxant effect were identical and comparable with the potency of GQV-NH2for causing a contractile effect in the LM. However, the potencies of the PAR4-APs in the RA and LM preparations were 20- to 150-fold lower than the potency of the receptor-selective PAR1-AP, TFLLR-NH2. We conclude that the PAR4-APs do not activate either PAR1or PAR2,and we suggest that along with PAR1and PAR2, PAR4may also be present in rat vascular and gastric smooth muscle.Key words: proteinase-activated receptors, PAR4, calcium, vascular smooth muscle, gastric smooth muscle, thrombin.