Aging is associated with vascular dysfunction, but its effects on the function of the vascular cells of coronary resistance arteries remain to be determined. This study aimed at evaluating the influence of aging (youth, young adulthood and middle age) on the activity of the endothelial (EC) and smooth muscle cells (SMC) of coronary arterioles. 3-, 6- and 11-month old male Wistar rats were anaesthetised deeply with sodium pentobarbital (30 mg/kg) and their heart were perfused according to the Langendorff procedure with a Krebs-Heinselett buffer containing glucose 11 mM at 37°C. The endothelial-dependent and -independent vasodilatations were then determined in the beating heart with acetylcholine (injections of 4, 10, 20, 40, 60, 80 and 100 pmoles) and nitroprusside (injections of 100, 200, 400, 600, 800 and 1000 pmoles) after vasoconstriction through a constant infusion of a thromboxane A2 analogue (U46619, 30 nM). The mitochondrial oxidative stress was then evaluated either in the whole heart by measuring the aconitase to fumarase ratio or in the isolated cardiac mitochondria by estimating the Amplex red-related rate of H2O2 production. Values are means ± S.E.M., compared by ANOVA. Although aging modified only minimally the cardiac mechanical function, it decreased progressively the endothelial-dependent vasodilatation (22.6 ± 0.8, 17.1 ± 1.5 and 13.4 ± 1.9% at 3, 6 and 11 months for the highest dose of acetylcholine, p < 0.01, n = 8). This was associated with specific age-associated modulation of EC and SMC functions. Compared to 3-month old rats, 6-month old animals had a higher endothelial vasodilatation capacity (553 ± 146 vs 108 ± 47 pmoles nitroprusside equivalents), but a lower SMC sensitivity to nitric oxide (28.6 ± 3.1 vs 53.6 ± 5.4%). This was not associated with any change of the oxidative stress. However, at middle age, the endothelial vasodilatation capacities returned to a normal value (186 ± 42 pmoles nitroprusside equivalents) and the SMC sensitivity to nitric oxide remained low (31.8 ± 2.1%). The reduction of the endothelial vasodilatation capacities between adulthood and middle age was associated with the development of a mitochondrial oxidative stress observed at the level of the isolated organelles and in the whole heart (the aconitase to fumarase ratio equalled 0.287 ± 0.012, 0.268 ± 0.023 and 0.157 ± 0.015 at 3, 6 and 11 months, respectively, p < 0.001, n=8). In conclusion, the SMC seem to be the first vascular cells whose function is deteriorated with aging. In contrast, the activity of EC appears to be improved until young adulthood and decreases thereafter with the development of a mitochondrial oxidative stress.