The effect of inhibition of the malate-aspartate shuttle on the cytoplasmic NADH/NAD ratio and NADH redox state and its corresponding effects on mitochondrial energetics in vascular smooth muscle were examined. Incubation of porcine carotid arteries with 0. 4 mmol amino-oxyacetic acid an inhibitor of glutamate-oxaloacetate transaminase and, hence the malate-aspartate shuttle, inhibited O2 consumption by 21%, decreased the content of phosphocreatine and inhibited activity of the tricarboxylic acid cycle. The rate of glycolysis and lactate production was increased but glucose oxidation was inhibited. These effects of amino-oxyacetic acid were accompanied by evidence of inhibition of the malate-aspartate shuttle and elevation in the cytoplasmic redox potential and NADH/NAD ratio as indicated by elevation of the concentration ratios of the lactate/pyruvate and glycerol-3-phosphate/dihydroxyacetone phosphate metabolite redox couples. Addition of the fatty acid octanoate normalized the adverse energetic effects of malate-aspartate shuttle inhibition. It is concluded that the malate-aspartate shuttle is a primary mode of clearance of NADH reducing equivalents from the cytoplasm in vascular smooth muscle. Glucose oxidation and lactate production are influenced by the activity of the shuttle. The results support the hypothesis that an increased cytoplasmic NADH redox potential impairs mitochondrial energy metabolism.