A sensitive method for the measurement of the ATP production rate in isolated skeletal muscle mitochondria is presented. Mitochondrial suspensions were prepared by differential centrifugation from approximately 80 mg of soleus muscle. ATP production rates were measured luminometrically, utilizing a reagent based upon firefly luciferase, which emits light proportional to the ATP concentration. In a group of 10 rats the ATP production rates were measured with the following substrate combinations: pyruvate + malate, palmitoyl-L-carnitine + malate, alpha-ketoglutarate, succinate + rotenone and succinate alone. The variance of the method including tissue preparation, protein determination and the luminometric determination of ATP production was estimated to be 10-14% for the various substrates. Compared to values in the literature, the present results show a good agreement for the substrates pyruvate + malate and palmitoyl-L-carnitine + malate, but lower rates were obtained in our study for alpha-ketoglutarate and succinate + rotenone. The advantage of the luminometric method is its high sensitivity. Only 30-40 mg of tissue is required for a complete determination, compared to 1-2 g for a similar assay of oxygen consumption. The method is intended for use in human subjects and will facilitate studies of mitochondrial respiration both in patients of different age groups and in healthy subjects.