Under anoxic incubation conditions heart cell cultures showed enhanced uptake of [U-14C]palmitic acid into neutral lipids, while incorporation into phospholipids was unaltered. Fractionation of the neutral lipids showed greatest incorporation of radiolabel into the triglyceride fraction. Uptake of fatty acid in normoxic cultures may be dependent upon the supply of glycerol 3-phosphate from glycolysis, as 2-deoxyglucose and L-lactate, respectively, inhibited and stimulated incorporation of fatty acid into neutral lipid fractions. When previously anoxic cultures were reoxygenated, oxidation of fatty acid was depressed and the mitochondrial function of anoxic cultures appeared to be more readily uncoupled by 2,4-dinitrophenol, in comparison with cultures maintained under normoxic conditions. Similar behaviour was seen when oxidation of endogenous lipid or oxidation of glucose was examined. Previously anoxic cultures show a preference for oxidation of endogenous rather than exogenous lipid substrates. The results suggest that anoxia-stimulated lipid accumulation may prove injurious to subsequent mitochondrial function and may be a contributory factor in the pathological processes associated with hypoxic injury of cardiac tissue.