The interstitial cells of Cajal (ICCs) are pacemakers in the gastrointestinal tract. The possibility of whether imatinib mesylate, a Kit receptor tyrosine kinase inhibitor, modulates pacemaker activities in the ICC was examined using the whole cell patch clamp technique. Imatinib decreased the amplitude of pacemaker potentials in a dose-dependent manner in current-clamp mode. Because the effects of imatinib on pacemaker potentials were the same as those of pinacidil, we examined the effect of glibenclamide on ICC exposed to imatinib. The effects of imatinib on pacemaker potentials were blocked by glibenclamide. To see whether the production of prostaglandins (PGs) is involved in the inhibitory effect of imatinib on pacemaker potentials, we tested the effects of naproxen (a non-selective cyclooxygenase inhibitor) and AH6809 (a prostaglandin EP1 and EP2 receptor antagonist). Naproxen and AH6809 blocked the inhibitory effects of imatinib on ICC. Butaprost (an EP2 receptor agonist) showed the actions on pacemaker potentials in the same manner as imatinib. However, SC 19220 (an EP1 receptor antagonist) has no effects. To investigate the involvement of cAMP and protein kinase A (PKA) in the effects of imatinib on ICC, SQ 22536 (an inhibitor of adenylate cyclase) and mPKAI (an inhibitor of myristoylated PKA) were used. Both SQ-22536 and mPKAI blocked the imatinib-mediated inhibition of pacemaker potentials. However, the protein kinase C (PKC) inhibitors did not block the imatinib-mediated inhibition of pacemaker potentials. These results indicate that imatinib inhibits the pacemaker potentials of ICC by activating ATP-sensitive K⁺ channels and PKA-dependent, PKC-independent manner.