The aim of the present investigation was to evaluate the possible role of arachidonic acid and other free fatty acids in ischemia-induced mitochondrial dysfunction. Respiratory activities were measured in mitochondria isolated from rat brains subjected to 15-30 min of decapitation ischemia. Addition of bovine serum albumin (BSA) to the mitochondria, isolated in BSA-free media, abolished an ischemia-induced increase in substrate-stimulated (state 4) respiration but only partly reversed a marked inhibition of substrate-, phosphate-, and ADP-stimulated (state 3) respiration caused by the ischemia. Individual free fatty acids were measured in aliquots of the same mitochondrial preparations before and after treatment with BSA. There was a significant increase in arachidonic (20:4), stearic (18:0), palmitic (16:0), and docosahexaenoic (22:6) acid during ischemia. BSA treatment removed all 20:4 and reduced the amount of 18:0 and 16:0, but had no significant effect on 22:6. The main conclusions were 1) that 20:4, 18:0, and 16.0 were responsible for the partial uncoupling (increase in state 4 respiration) of mitochondrial respiration during ischemia, 2) that the inhibition of state 3 respiration caused by ischemia could only partly be attributed to an effect of FFAs, and 3) that the ischemia-induced mitochondrial dysfunction was caused by a combination of factors including 20:4.