The mitochondrial oxidation of fatty acids (FAO) is the main energy-producing pathway in skeletal and cardiac muscle. Starting from standard muscle biopsies (100-200 mg), we determined the optimal conditions of mitochondrial oxygen consumption by the FAO pathway, and in parallel we performed the isolation and primary culture of muscle cells to test their cellular FAO capacities. The determinations of maximal b-oxidation rates in the presence of palmitoyl-CoA or palmitoyl-L L-carnitine (mean AE SEM: 32:5 AE 2:0 and 34:1 AE 1:3 nmol O 2 min À1 mg À1 protein, n ¼ 16, respectively) provide a screening method of mitochondrial fatty acid transport system and intra-mitochondrial b-oxidation. We also determined the conditions of tritiated palmitate oxidation by human myoblasts (mean AE SEM: 6:6 AE 0:1 nmol 3 H fatty acid h À1 mg À1 protein, n ¼ 8), and show that b-oxidation defects can be detected in our experiments. Overall, we propose an original laboratory test to investigate FAO in human skeletal muscle and to screen for FAO disorders in myopathies and cardiomyopathies in human.