Bertocci, Loren A., John G. Jones, Craig R. Malloy, Ronald G. Victor, and Gail D. Thomas. Oxidation of lactate and acetate in rat skeletal muscle: analysis by ¹³C-nuclear magnetic resonance spectroscopy. J. Appl. Physiol. 83(1): 32-39, 1997.The balance between carbohydrate and fatty acid utilization in skeletal muscle previously has been studied in vivo by using a variety of methods such as arteriovenous concentration differences and radioactive isotope tracer techniques. However, these methodologies provide only indirect estimates of substrate oxidation. We used ¹³C-nuclear magnetic resonance (NMR) spectroscopy and non-steady-state isotopomer analysis to directly quantify the relative oxidation of two competing exogenous substrates in rat skeletal muscles. We infused [1,2-¹³C]acetate and [3-¹³C]lactate intravenously in anesthetized rats during the final 30 min of 35 (n = 10) or 95 (n = 10) min of intense, unilateral, rhythmic hindlimb contractions. ¹³C-NMR spectroscopy and isotopomer analysis were performed on extracts of gastrocnemius and soleus muscles from both the contracting and contralateral resting hindlimbs. We found that 1) [¹³C]lactate and [¹³C]acetate were taken up and oxidized by both resting and contracting skeletal muscles; and 2) high-intensity muscle contractions altered the pattern of substrate utilization such that the relative oxidation of acetate decreased while that of lactate remained unchanged or increased. Based on these findings, we propose that ¹³C-NMR spectroscopy in combination with isotopomer analysis can be used to study the general dynamics of substrate competition between carbohydrates and fats in rat skeletal muscle.