Mitonchondria isolated from the Morris hepatoma 7777 demonstrated a markedly different phospholipid composition from those of control mitochondria, both with respect to the amounts of the various types present and the fatty acid composition. The level of polyunsaturated fatty acids in the mitochondrial phospholipids was lowered, wheras there was an increase in the level of monounsaturated fatty acids. Moreover, the usual distribution of saturated fatty acids at position 1 and polyunsaturated fatty acids at position 2 does not exist in hepatoma phospholipids; a high percentage of monounsaturated fatty acids was found at both positions. The cardiolipin content was lower in hepatoma mitochondria (3.7%) than in livers of animals with hepatomas (5.2%). There was, however, some compensation in the amount of acidic phospholipids in these mitochondria due to an increase in phosphatidylserine (4.9% versus 1.3%). The force-area curves of the hepatoma phospholipids spread on a monomolecular film demonstrated a smaller area per molecule than those from liver mitochondria. The zeta potential of liposomes of the hepatoma phospholipids (-45) was less than those of control mitochondria (-81), as determined by microelectrophoresis. The calcium-stimulated phospholipase A activity of the hepatoma mitochondria appeared to be more readily expressed than the same activity in liver organelles. The maximal activity was lower, however, than that noted in liver mitochondria. Furthermore, by following the incorporation of [3H]ethanolamine into mitochondria phospholipids, it was established that the conversion of glycerophosphorylethanolamine to glycerophosphorylcholine was increased in the hepatoma. These observations suggest dramatic changes in phospholipid metabolism in the hepatoma, at the level of both the endoplasmic reticulum and the mitochondrion. Accompanying the changes in phospholipid compositon and metabolism were alterations in mitochondrial energy-linked processes. The hepatoma mitochondria demonstrated lower respiratory control ratios even when isolated in an isotonic solution containing 1mM ethylenediaminetetraacetate and bovine serum albumin (0.5 mg/ml). This was due to increased state 4 respiration.