Abstract: AIM: To investigate the protective effect of hypotonicity that, against ischemic heart, may be activating KATP channel. METHODS: RT-PCR technique was used to detect the expression of Kir (inward rectifier potassium channel) genes in rat cardiac myocytes. Single-channel patch clamp technique was used to record the volume-sensitive potassium current of acutely isolated rat cardiac myocytes under hypotonic condition. Rat hearts were divided into five groups: 1) ischemia (CON) group, 2) glibenclamide (Glib) group, 3) ischemic preconditioning (IPC) group, 4) hypotonic solution treatment (HYPO) group and 5) hypotonic solution plus glibenclamide treatment (HYPO+Glib) group. Isolated rat hearts were Langendorff perfused and cardiac function was assessed by measuring the LVDP (left ventricular developed pressure) and ±LVdP/dtmax (maximal rate of left ventricular pressure development) to observe the protection effect of hypotonic solution to ischemic heart. RESULTS: Both Kir6.2 and Kir6.1 were expressed in the rat heart. In single cardiac myocytes, hypotonic solution activated an ATP-sensitive potassium (KATP) current with the single-channel conductance of (30.34±1.76) pS at 80 mV and the open probability (Po) increased from 0.01±0.01 to 0.51±0.12. The current was blocked by KATP channel blocker glibenclamide (10 μmol/L). Pre-perfusing the heart with hypotonic solution for 5 min before ischemia/reperfusion resulted in a significant improvement in heart function compared with that in CON group ［at 60 min of reperfusion, LVDP recovered from (45.02±1.70)% to (58.43±0.86)%, +LVdP/dtmax recovered from (42.85±4.45)% to (61.96±2.98)% and -LVdP/dtmax recovered from (53.94±2.52)% to (66.60±3.86)%; P<0.05］. The cardiac protective effect of hypotonicity was eliminated by Glib. CONCLUSION: KATP channels in rat cardiomyocytes have volume sensitivity and hypotonicity may contribute to the cardioprotection during ischemia/reperfusion injury by activating KATP channels.