The availability of genetically modified mice requires the development of methods to assess heart function and metabolism in the intact beating organ. Using radioactive substrates and ex vivo perfusion of the mouse heart in the working mode, previous studies have documented glucose and fatty acid oxidation pathways. This study was aimed at characterizing the metabolism of other potentially important exogenous carbohydrate sources, namely lactate and pyruvate. This was achieved using ¹³C-methods. The mouse heart perfusion setup and buffer composition were optimized to reproduce conditions close to the in vivo milieu, in terms of workload, cardiac functions and substrate/hormone supply to the heart (11mM glucose, 0.8nM insulin, 50µM carnitine, 1.5mM lactate, 0.2mM pyruvate, 5nM epinephrine, 0.7mM oleate and 3% albumin). The use of three differentially ¹³C-labeled carbohydrates and a ¹³C-long chain fatty acid allowed the quantitative assessment of the metabolic origin and fate of tissue pyruvate, as well as the relative contribution of substrates feeding acetyl-CoA (pyruvate and fatty acids) and oxaloacetate (pyruvate) for mitochondrial citrate synthesis. Beyond concurring with the notion that the mouse heart preferentially uses fatty acids for energy production (63.5 ± 3.9%) and regulates its fuel selection according to the Randle cycle, our study reports for the first time in the mouse heart the following findings. Firstly, lactate is the major carbohydrate contributing to pyruvate formation (42.0 ± 2.3%). Secondly, lactate and pyruvate are constantly being taken up and released by the heart, supporting the concept of compartmentation of lactate and glucose metabolism. Finally, mitochondrial anaplerotic pyruvate carboxylation and citrate efflux represent 4.9 ± 1.8% and 0.8 ± 0.1% of the citric acid cycle flux, respectively, and are modulated by substrate supply. The described ¹³C-strategy combined with an experimental setup that enables a continuous monitoring of physiological parameters, offers a unique model to clarify the link between metabolic alterations, cardiac dysfunction and disease development.