Recording simultaneously in vitro the changes of endoluminal pressure (index of circular muscle activity) and isometric tension (index of longitudinal muscle activity), we examined the mechanisms responsible for the apamin-sensitive relaxant and contractile responses induced by protease-activated receptor (PAR)-1 and PAR-2 activating peptides, SFLLRN-NH 2 and SLIGRL-NH 2 , respectively, in rat colon. In the circular muscle, the inhibitory effects of SFLLRN-NH 2 and SLIGRL-NH 2 were significantly reduced by ryanodine, an inhibitor of Ca 2ϩ release from the sarcoplasmic reticulum, but unaffected by 1-, a protein kinase C (PKC) inhibitor, or genistein, a tyrosine kinase inhibitor. In the longitudinal muscle, the contractile responses to SFLLRN-NH 2 and SLIGRL-NH 2 were significantly reduced by nifedipine, an L-type calcium channel blocker, ryanodine, GF109203X, genistein, and abolished by U73122. The effects of genistein were additive with GF109203X but not with nifedipine. In the longitudinal muscle, the relaxant responses to the highest concentrations of SFLLRN-NH 2 and SLIGRL-NH 2 were abolished by nifedipine, reduced by genistein, and unaffected by ryanodine or GF109203X. In conclusion, influx of extracellular Ca 2ϩ through L-type voltage-dependent channels or release of Ca 2ϩ from intracellular stores are determining for the opening of the apamin-sensitive K ϩ channels responsible for longitudinal muscle relaxation or circular muscle inhibitory response, respectively, in rat colon. The longitudinal muscle contraction is mediated by activation of PLC; PKC and tyrosine kinase are involved in the cascade process, playing a parallel role. Indeed, tyrosine kinase and L-type Ca 2ϩ channels would act sequentially.