We examined the mechanism by which protease-activated receptor (PAR)-1 is desensitized by comparing the effect of thrombin and the soluble agonist peptide SFLLRN on Ca2+responses in HSY-EA1 cells. Thrombin-induced increases in cytosolic Ca2+concentrations ([Ca2+]i) returned to basal levels within 60 s, but SFLLRN generated a sustained [Ca2+]ielevation. Interestingly, thrombin-desensitized cells partially retained their ability to respond to SFLLRN. We desensitized PAR-2 by pretreating cells with SLIGKV to confirm that this response was not due to PAR-2, which can recognize SFLLRN. The highly specific PAR-1 agonist peptide TFLLR also increased [Ca2+]iin PAR-2-desensitized cells pretreated with thrombin. These observations indicate that thrombin disarms PAR-1 from further proteolytic activation, but leaves the receptor responsive for non-tethered ligands.