AbstractThe protonmotive force (Δp) of muscle mitochondria was measured by estimating the distribution of14C‐labeled TPMP (trimethylphenylphosphonium iodide) and14C‐labeled acetate across the inner membrane of muscle mitochondria. The matrix volume was simultaneously determined using3H‐labeled H2O and3H‐labeled mannitol and repeated drying to distinguish the label in these 2 compounds. Rapid separation of mitochondria from the incubation medium by centrifugation through silicone oil avoids the problems of potential anaerobic conditions associated with conventional centrifugation and large volumes of trapped media associated with filtration. The value for Δp (mean ± SD) was 192 ± 26 mV in 30 determinations with rat muscle mitochondria during state 4. Measurement of oxygen consumption allowed calculation of membrane conductance (Cm, H+) which was 0.49 ± 0.18 nmol of H+/min/mg protein/mV. The values for δp and Cm, H+are reported for a variety of experimental conditions and are consistent with Mitchell's chemiosmotic theory. Biopsy specimens obtained from human muscle gave state‐4 Δp values of 197 ± 30 mV (n= 5) and Cm, H+values of 0.52 ± 0.12 nmol of H+/min/mg/mV (n= 4). This Δp assay is the first described for coupled mammalian muscle mitochondria and will be useful in assessin