Lipid composition of the mitochondria in Ehrlich Lettré Hyperdiploid (ELD) ascites tumor cells of Swiss mice was determined. The total lipid contained 19% neutral lipid and 81% phospholipid. The major neutral lipids were sterols (64% of neutral lipids); free cholesterol (44.5%), esterified cholesterol (15.7%) and other plant sterols (3.8%), such as campesterol (2.3%) and β-sitosterol (1.5%). Nonsterol neutral lipids were triglycerides (22.0%), diglycerides (2.9%), free fatty acids (8.8%), and CoQ (2.3%). Phospholipids were comprised of choline glycerophosphatides (48.9%) of lipid p), ethanolamine glycerophosphatides (27.3%), cardiolipin (11.3%), inositol glycerophosphatides (4.2%), sphingomyelin (6.4%), and lysolecithin (1.9%). Fatty acids in the neutral lipids were highly unsaturated; cholesterol esters (83%), triglycerides (64%), and free fatty acids (51%). However, fatty acids unsaturation varied among the different phospholipids; cardiolipin (72%), ethanolamine glycerophosphatides (59%), choline glycerophosphatides (55%), phosphatidyl inositol (52%), and sphingomyelin (19%). Carbon 18 fatty acids predominated (50 to 77%) in neutral lipids and phospholipids except for sphingomyelin (6%) and phosphatidyl inositol (37%). The acidic phospholipids, such as cardiolipin and phosphatidyl inositol of tumor mitochondria, was similar to that of comparable rodent liver mitochondria but the amount of cholesterol was strikingly greater than had been reported previously for normal mitochondria of various tissues.