Objective: Optimal timing of therapeutic hypothermia for cardiac ischemia is unknown. Our prior work suggests that ischemia with rapid reperfusion (I/R) in cardiomyocytes can be more damaging than prolonged ischemia alone. Also, these cardiomyocytes demonstrate protein kinase C (PKC) activation and nitric oxide (NO) signaling that confer protection against I/R injury. Thus, we hypothesized that hypothermia will protect most using extended ischemia and early reperfusion cooling, and is mediated via PKC and NO synthase (NOS). Methods: Chick cardiomyocytes were exposed to an established model of 1-h ischemia/3-h reperfusion, and the same field of initially contracting cells monitored for viability and NO generation. Results: Normothermic I/R resulted in 49.7 ± 3.4% cell death. Hypothermia induction to 25°C was most protective (14.3 ± 0.6% death, P < 0.001 vs. I/R control) when instituted during extended ischemia and early reperfusion, compared to induction after reperfusion (22.4 ± 2.9% death). Protection was completely lost if onset of cooling was delayed by 15 min of reperfusion (45.0 ± 8.2% death). Extended ischemia/early reperfusion cooling was associated with increased and sustained NO generation at reperfusion and decreased caspase 3 activation. The NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME) (200 µM) reversed these changes and attenuated hypothermia protection. In addition, the PKC inhibitor myr-PKC v1-2 (5 µM) also reversed NO production and hypothermia protection. Conclusion: Therapeutic hypothermia initiated during extended ischemia/early reperfusion optimally protects cardiomyocytes from I/R injury. Such protection is mediated by activation of PKC&#949; and NOS, leading to increased NO production at reperfusion.