Protease-activated receptor-1 (PAR1), PAR2 and PAR4 activation can alter the gastrointestinal motility. To investigate effects mediated by PARs in the lower esophageal sphincter, we measured contraction or relaxation of transverse strips from the guinea-pig lower esophageal sphincter caused by PAR1 (TFLLR-NH 2 and SFLLRN-NH 2 ), PAR2 (SLIGKV-NH 2 and SLIGRL-NH 2 ) and PAR4 peptide agonists (GYPGKF-NH 2 , GYPGQV-NH 2 and AYPGKF-NH 2 ) as well as PAR protease activators (thrombin and trypsin). In resting lower esophageal sphincter strips, TFLLR-NH 2 and SFLLRN-NH 2 caused moderate concentration-dependent relaxation whereas thrombin did not cause any relaxation or contraction. Furthermore, in carbachol-contracted strips, TFLLR-NH 2 and SFLLRN-NH 2 caused marked whereas thrombin caused mild concentration-dependent relaxation. These indicate the existence of PAR1 mediating relaxation. Similarly, in resting lower esophageal sphincter strips, trypsin caused moderate concentration-dependent relaxation whereas SLIGRL-NH 2 and SLIGKV-NH 2 did not cause any relaxation or contraction. In addition, in carbachol-contracted strips, trypsin caused marked whereas SLIGRL-NH 2 and SLIGKV-NH 2 caused mild concentration-dependent relaxation. These indicate the existence of PAR2 mediating relaxation. The relaxant response of thrombin, TFLLR-NH 2 , trypsin and SLIGKV-NH 2 was insensitive to atropine or tetrodotoxin, suggesting a direct effect. The relaxant response of trypsin was not affected by apamin, charybdotoxin, indomethacin and capsaicin but was attenuated by N G -nitro-l-arginine methyl ester, indicating involvement of NO. FSLLR-NH 2 , a PAR1 control peptide, and VKGILS-NH 2 , a PAR2 control peptide, as well as all three PAR4 peptide agonists, GYPGKF-NH 2 , GYPGQV-NH 2 and AYPGKF-NH 2 , did not cause any relaxation or contraction. Taken together, these results demonstrate that PAR1 and PAR2 but not PAR4 mediate relaxations in the guinea-pig lower esophageal sphincter.