AbstractPreliminary experiments with 2,4‐dinitrophenol (DNP), 2,4,6‐trinitrophenol (TNP), and sodium azide (NaN3) indicated that most of the oxygen consumption by ribbed mussel gill tissue is the result of mitochondrial respiration. A procedure utilizing isoosmotic sucrose, EGTA, defatted serum albumin, and HEPES as the isolation medium was devised for the preparation of fully coupled ribbed mussel gill mitochondria. Optimal rates of respiration and respiratory coupling required substrate, ADP, inorganic phosphate, and a fairly high KCl concentration (90 mM) in the assay medium. Glutamate, proline, malate, and succinate stimulated oxygen consumption with high respiratory control indices and P/O of 3, 3, 3 and 2, respectively. Pyruvate was a weak stimulator of mitochondrial respiration and showed a low respiratory control index with a low P/O. Preparation of gill mitochondria in isoosmotic solutions containing high KCl concentrations (150 mM) yielded mitochondria showing state 2 respiration, slow partially uncoupled ATP synthesis during state 3 respiration and no state 4 respiration. D‐mannitol was not used in the mitochondrial isolation or assay medium because of the probable presence of a D‐mannitol oxidase in these gill mitoc