The objective of this study was to examine the role of chloride (Cl-) channels in the myocardial protection of ischemic preconditioning (IP). Isolated rabbit ventricular myocytes were preconditioned with 10-minute simulated ischemia (SI) and 20-minute simulated reperfusion (SR) or not preconditioned (control). The myocytes then received 180-minute SI or 45-minute SI/120-minute SR. Indanyloxyacetic acid 94 (IAA-94, 10 micromol/L) or 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 1 micromol/L) was administered before IP or before SI or SI/SR to inhibit Cl- channels. Electrophysiological studies indicate that these drugs, at the concentrations used, selectively abolished Cl- currents activated under hypo-osmotic conditions (215 versus 290 mOsm). IP significantly (P<0.001) reduced the percentage of dead myocytes after 60-minute (30.8+/-1.3%, mean+/-SEM), 90-minute (35.3+/-1.3%), and 120-minute (39.2+/-1.7%) SI compared with controls (44.7+/-1.6%, 54.5+/-1.3%, and 58.9+/-1.8%, respectively) and after 45-minute SI/120-minute SR (36.3+/-0.6%) compared with control (56.6+/-2.2%). Hypo-osmotic stress also produced protection similar to IP. IAA-94 or NPPB abolished the protection of both IP and hypo-osmotic stress. In buffer-perfused rabbit hearts preconditioned with three 5-minute ischemia/10-minute reperfusion cycles given before the 40-minute long ischemia and 60-minute reperfusion, IP significantly (P<0.0001) reduced infarct size (IP+vehicle, 4.7+/-0.9%, versus control+vehicle, 26.6+/-3.3%; mean+/-SEM). Again, IAA-94 or NPPB abolished the protection of IP. Our results implicate Cl- channels in the IP protection of the myocardium against ischemic/reperfusion injury and demonstrate that hypo-osmotic stress is capable of preconditioning cardiomyocytes.