OBJECTIVE To explore the late cardioprotection induced by morphine preconditioning and determine the role of inducible nitric oxide synthase (iNOS) in mediating this effect. METHODS Thirty-two wild type (WT) mice and 16 iNOS gene knockout mice, totally 48 mice, underwent ligation of the left anterior descending coronary artery (LAD) for 45 minutes and reperfusion for 120 minutes. The 32 wild type mice were randomly divided into 4 groups of 8 mice: WT control group, 24 hours before the heart occlusion, normal saline was given; WT + morphine group, 24 hours before the heart occlusion morphine was administered; WT + SMT group, 24 hours before the heart occlusion normal saline was administered and 30 minutes before heart occlusion S-methylthiourea sulfate (SMT), a selective inhibitor of iNOS, was administered; and WT + morphine + SMT group, 24 hours before the heart occlusion morphine was administered and 30 minutes before heart occlusion SMT was administered. The 16 iNOS gene knockout mice were randomly divided into 2 groups of 8 mice: iNOS (-/-) control group (24 hours before the heart occlusion normal saline was given) and iNOS (-/-) + morphine group (24 hours before heart occlusion. morphine was administered). One hundred and twenty minutes after reperfusion, the LAD was re-ligated at the original site for all the mice. Evans blue was injected via right carotid artery catheterization to stain the non-ischemic area of the heart. Then the hearts of all mice were taken out, cut into 5 pieces with similar thickness, and put into the solution of 2, 3, 5-triphenyltetrazolium chloride (TTC). The heart was fixed in formalin solution, underwent digital photography, and weighed. NIH Image software was used to calculate the area of the left ventricle (LV), infarct size (IS), and area at risk (AAR). The size of myocardial ischemia was expressed as AAR/LV, and the scope of myocardial infarction was expressed as IS/AAR. RESULTS The IS/AAR of the WT control group was 43% +/- 5%, significantly larger than that of the WT + morphine group (22% +/- 4% P < 0.05). The value of IS/AAR of the WT + morphine + SMI group was 43% +/- 4%, not significantly different from that of the WT control group (P > 0.05). The IS/AAR of the iNOS (-/-) control group was 44% +/- 4%, also not significantly different from that of the WT control group (P > 0.05). The IS/AAR of the iNOS (-/-) + morphine group was 42% +/- 5%, not significantly different from that of the iNOS (-/-) control group (P > 0.05). CONCLUSIONS Morphine preconditioning induces late cardioprotection in mice via an iNOS-dependent pathway. Pretreatment with SMT abolishes the morphine-induced reduction of infarct size. In addition, morphine fails to reduce infarct size in iNOS gene knockout mice.