Exposure of rat liver to periods of ischemia results in a progressive increase in mitochondrial free fatty acids (FFA). The levels increase some 6-7-fold when the liver has been ischemic either for 2 hr at 38 ° or 13 hr at 24 °. A decline in respiratory control (rate of O2 uptake with ADP/rate of O2 uptake without ADP) parallels the rise in FFA at both temperatures. Significant depression of respiratory control is observed when comparable amounts of FFA are added to normal mitochondria. Bovine serum albumin (BSA), which restores respiratory control to a considerable degree and enhances the stability of mitochondria isolated from ischemic tissue, reduces the FFA levels about 70%. Despite the presence of BSA during isolation and testing of the mitochondria, there is still a gradual decline in the respiratory control ratio over a period of ischemia lasting several hours, primarily because of a diminution in the state 3 rate of respiration. The levels of mitochondrial phosphatidyl ethanolamine decreased somewhat, but the percentage change was small. Phosphatidyl choline remained essentially unchanged. A more significant decrease in cardiolipin was observed. Part of this change must be ascribed to dilution of the mitochondria by increased microsomal contamination. The data suggest that mitochondrial changes during ischemia involve (a) a BSA-reversible phase produced by FFA released by lipolysis, and (b) a further change, not reversed by BSA, which may be due to loss of essential mitochondrial lipid like cardiolipin, the presence of lysophosphatides, or proteolysis. Morphological changes in mitochondria in liver sections are consistent with these interpretations.