The purpose of this study was to determine the contribution of long-chain fatty acids relative to other mitochondrial substrates in active vascular smooth muscle (VSM). Hog carotid arteries were isometrically contracted in physiological saline solution containing 0.71 m M U-<sup>13</sup>C-oleic acid (bound to albumin at a ratio of 6.8:1), 5 m M 1-<sup>13</sup>C-glucose and 1 m M acetate in the presence or absence of 5 m M carnitine for 6 h at 37°C. Substrate oxidation was determined using <sup>13</sup>C-isotopomer analysis of glutamate. Although oxidation of oleic acid could not be measured at physiological concentrations [0.5 m M (1:1)], oleic acid oxidation was approximately 5% of the total substrates oxidized at the higher concentration examined. Although insignificant, carnitine increased oleic acid oxidation to approximately 8%, and resulted in a decrease in endogenous lipid oxidation, which was 2–12% of the total substrates oxidized. Oxidation of glucose and acetate did not significantly change due to the inclusion of oleic acid in the incubation solutions. Therefore, we conclude that exogenous long-chain fatty acids are minor contributors to substrate oxidation (approximately 5%) in VSM compared to other mitochondrial substrates, such as glucose and acetate, which account for approximately 80% of the substrates oxidized by VSM.