Previously we showed that Brown Norway (BN/Mcw) rats are more resistant to myocardial ischemia-reperfusion (I/R) injury than Dahl S (SS/Mcw) rats due to increased nitric oxide ( ·NO) generation secondary to increased heat shock protein 90 (HSP90) association with endothelial nitric oxide synthase (NOS3). Here we determined whether increased resistance to I/R injury in BN/Mcw hearts is also related to tetrahydrobiopterin (BH₄) and GTP cyclohydrolase I (GCH-1), the rate-limiting enzyme for BH₄ synthesis. We observed that BH₄ supplementation via sepiapterin (SP) and inhibition of GCH-1 via 2,4-diamino-6-hydroxypyrimidine (DAHP) differentially modulate cardioprotection and that SP alters the association of HSP90 with NOS3. BH₄ levels were significantly higher and 7,8-dihydrobiopterin (BH₂) levels were significantly lower in BN/Mcw than in SS/Mcw hearts. The BH₄-to-BH₂ ratio in BN/ Mcw was more than two times that in SS/Mcw hearts. After I/R, BH₄ decreased and BH₂ increased in hearts from both strains compared with their preischemia levels. However, the increase in BH₂ in SS/Mcw hearts was significantly higher than in BN/Mcw hearts. Real-time PCR revealed that BN/Mcw hearts contained more GCH-1 transcripts than SS/Mcw hearts. SP increased recovery of left ventricular developed pressure (rLVDP) following I/R as well as decreased superoxide (O₂^.-) and increased · NO in SS/Mcw hearts but not in BN/Mcw hearts. DAHP decreased rLVDP as well as increased O₂^.- and decreased · NO in BN/Mcw hearts compared with controls but not in SS/Mcw hearts. SP increased the association of HSP90 with NOS3. These data indicate that BH₄ mediates resistance to I/R by acting as a cofactor and enhancing HSP90-NOS3 association. Copyright © 2009 the American Physiological Society.