OBJECTIVE To explore the relationship between endothelial nitric oxide synthase (eNOS) activity, insulin resistance and macrovascular disease in patients with acute myocardial infarction (AMI). METHODS AMI patients were grouped according to the presence (group A, n = 49) or absence (group B, n = 48) of macrovascular disease. A healthy control group was also recruited (group C, n = 43). eNOS activity and nitric oxide (NO), endothelin-1 (ET-1), fasting plasma glucose and fasting insulin levels were compared across groups. The homeostasis model assessment of insulin resistance (HOMA- IR) was calculated in each participant and correlations between biochemical parameters were determined. RESULTS eNOS and NO levels were significantly lower in group A compared with the other groups. Conversely, ET-1 levels and the HOMA-IR were significantly higher in group A. eNOS activity and NO levels were significantly lower, and ET-1 levels and HOMA-IR were significantly higher, in group B compared with controls. Across the groups there were inverse correlations in AMI patients between eNOS and HOMAIR, NO and HOMA-IR, eNOS and ET-1, and NO and ET-1, and positive correlations between eNOS and NO, regardless of whether macrovascular disease was present. CONCLUSIONS There is a close relationship between eNOS activity and the development of insulin resistance and macrovascular disease in AMI patients.