1. In this study, the efficiency of energy conversion in skeletal muscles from the mouse was determined before and after a series of contractions that produced a moderate level of fatigue. 2. Initial mechanical efficiency was defined as the ratio of mechanical power output to the rate of initial enthalpy output. The rate of initial enthalpy output was the sum of the power output and rate of initial heat output. Heat output was measured using a thermopile with high temporal resolution. 3. Experiments were performed in vitro (25 degrees C) using bundles of fibres from fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus muscles from mice. Muscles were fatigued using a series of thirty isometric tetani. Initial mechanical efficiency was determined before and again immediately after the fatigue protocol using a series of isovelocity contractions at shortening velocities between 0 and the maximum shortening velocity (Vmax). Efficiency was determined over the second half of the shortening at each velocity. 4. The fatigue protocol significantly reduced maximum isometric force Vmax, maximum power output and flattened the force-velocity curve. The magnitude of these effects was greater in EDL muscle than soleus muscle. In unfatigued muscle, the maximum mechanical efficiency was 0.333 for EDL muscles and 0.425 for soleus muscles. In both muscle types, the fatiguing contractions caused maximum efficiency to decrease. The magnitude of the decrease was 15% of the pre-fatigue value in EDL and 9% in soleus. 5. In a separate series of experiments, the effect of the fatigue protocol on the partitioning of energy expenditure between crossbridge and non-crossbridge sources was determined. Data from these experiments enabled the efficiency of energy conversion by the crossbridges to be estimated. It was concluded that the decrease in initial mechanical efficiency reflected a decrease in the efficiency of energy conversion by the crossbridges.