Isolated mitochondria were added to the assay media and oxygen consumption was measured after the addition of malate and pyruvate (mal+pyr), adenosine diphosphate (ADP), glutamate (Glu), succinate (Suc), and rotenone (Rot). Robust oxygen consumption due to complex I (CI) and complex II (CII) was evident in wild type mitochondria, while only complex II activity was present in ACAD9−/− mitochondria.