BACKGROUND: The molecular mechanisms of exercise training induced cardiovascular protection are poorly understood. There is growing evidence that reactive oxygen species may be involved in a number of these adaptations and that antioxidants may be used to investigate this effect. OBJECTIVE: To determine the effects of exercise training and/or antioxidant supplementation on myocardial endothelium and vascular endothelium gene expression. METHODS: Male Wistar rats were divided into four groups: i) control; ii) exercise trained (90 min of treadmill running 4d per week, 14 weeks); iii) antioxidant-supplemented (α-tocopherol 1000 IU kg(-1) diet and α-lipoic acid 1.6 g kg(-1) diet, mixed with rat chow) and iv) exercise trained and antioxidant-supplemented. RESULTS: cDNA microarray analysis showed diverse expression changes in both left ventricular and coronary artery endothelial cells. In particular, RT-PCR analysis showed that a gene involved in cardiovascular disease progression, Ras homolog gene family member A, was down-regulated by exercise, and up-regulated by antioxidant supplementation in left ventricular endothelial cells. Furthermore, an important gene involved in inflammation, IL-6, was down-regulated by all treatments. CONCLUSIONS: Exercise training and/or antioxidant supplementation affects cardiac endothelial cell gene expression, and their effects on genes such as ras homolog gene family member A and IL-6 provides insight into the molecular mechanisms of their influences on cardiovascular diseases.