Glutamine, the most abundant amino acid in plasma, has attracted considerable interest for its cardioprotective properties. The primary effect of glutamine in the heart is commonly believed to be mediated via its anaplerotic metabolism to citric acid cycle (CAC) intermediates; however, there is little direct evidence to support this concept. Another potential candidate is the hexosamine biosynthetic pathway (HBP), which has recently been shown to modulate cardiomyocyte function and metabolism. Therefore, the goal of this study was to evaluate the contribution of anaplerosis and the HBP to the acute metabolic effects of glutamine in the heart. Normoxic ex vivo working rat hearts were perfused with 13C-labeled substrates to assess relevant metabolic fluxes either with a physiological mixture of carbohydrates and a fatty acid (control) or under conditions of restricted pyruvate anaplerosis. Addition of a physiological concentration of glutamine (0.5mM) had no effect on contractile function of hearts perfused under the control condition, but improved that of hearts perfused under restricted pyruvate anaplerosis. Changes in CAC intermediate concentrations as well as 13C-enrichment from [U-13C]glutamine did not support a major role of glutamine anaplerosis under any conditions. Under the control condition, however, glutamine significantly increased the contribution of exogenous oleate to β-oxidation, 1.6-fold, and triglyceride formation, 2.8-fold. Glutamine had no effect on malonyl-CoA or AMP kinase activity levels; however, it resulted in a higher plasma membrane level of the fatty acid transporter CD36. These metabolic effects of glutamine were reversed by azaserine, which inhibits glucose entry into the HPB. Our results reveal a metabolic role of physiological concentration of glutamine in the healthy working heart beyond anaplerosis. This role appears to involve the HBP and regulation of fatty acid entry and metabolism via CD36. This article is part of a Special Issue entitled "Focus on Cardiac Metabolism". © 2012 Elsevier Ltd.