The present investigation was designed to examine the effects of free arachidonic acid (20:4), in concentrations relevant to cerebral ischemia, on brain mitochondrial respiratory activities and the reversibility of these effects. Incubation of brain mitochondria with 20:4 caused a dose-dependent increase in substratesupported (state 4) respiration (i.e., uncoupling) and a concomitant inhibition of substrate-, phosphate-, and ADP-supported (state 3) or dinitrophenol-supported state (3u) respiration. The temperature dependence of the 20:4 effects on mitochondrial respiration was also studied. It was found that the uncoupling and the respiratory inhibition were at least as pronounced at physiological temperatures as at room temperature. Arrhenius plots of the state 3 respiratory rates suggested that 20:4 did not cause a significant change in membrane fluidity. Addition of bovine serum albumin to the reaction medium following preincubation with 20:4 reversed the uncoupling effect but only partly reversed the inhibition of state 3 respiration. The results suggest 1) that 20:4 may inhibit mitochondrial ATP production during conditions of incomplete cerebral ischemia and 2) that 20:4 may limit the postischemic recovery of mitochondrial function.