NAD+-linked isocitrate dehydrogenase was found in the brain, heart, gills, kidney, liver and muscle of trout, and in the liver and muscle of eel. A complex homogenization buffer containing 1 mM ADP, 5 mM MgSO4, 5 mM citrate and 40% glycerol is required for retrieval of significant amounts of stable enzyme. The highest activities were found in brain of trout and the lowest in white muscle of trout and eel. The enzyme was partially purified from frozen trout heart to a final activity of 0.04 μM/min/mg protein, and the kinetic properties of this partially purified enzyme were studied. The enzyme requires either Mn2+ or Mg2+ for activity, higher activities being observed with Mn2+. Saturation kinetics for DL-isocitrate were sigmoidal, apparent S0·5=8.2±0.6 mM and nH=1.8±0.2, in the absence of ADP, changing to hyperbolic, apparent S0·5=1.4±0.3 mM and nH=1.0, with 1 mM ADP added. Citrate and Ca2+ were found to activate the enzyme to a small extent. NADH strongly inhibited the enzyme, I50=3.7±0.5 μM. ATP was also found to be an inhibitor, I50=7.2±1.4 mM. These properties are consistent with the role of the enzyme as a major control site of the tricarboxylic acid cycle.