The aim of this study was to make cellular-level measurements of the mechanical efficiency of mouse cardiac muscle and to use these measurements to determine (1) the work performed by a cross-bridge in one ATP-splitting cycle and (2) the fraction of the free energy available in metabolic substrates that is transferred by oxidative phosphorylation to free energy in ATP (i.e. mitochondrial thermodynamic efficiency). Experiments were performed using isolated left ventricular mouse papillary muscles (n = 9; studied at 27°C) and the myothermic technique. The production of work and heat was measured during and after 40 contractions at a contraction frequency of 2 Hz. Each contraction consisted of a brief isometric period followed by isovelocity shortening. Work output, heat output and enthalpy output were all independent of shortening velocity. Maximum initial mechanical efficiency (mean ± SEM) was 31.1 ± 1.3% and maximum net mechanical efficiency 16.9 ± 1.5%. It was calculated that the maximum work per cross-bridge cycle was 20 zJ, comparable to values for mouse skeletal muscle, and that mitochondrial thermodynamic efficiency was 72%. Analysis of data in the literature suggests that mitochondrial efficiency of cardiac muscle from other species is also likely to be between 70 and 80%.