The yields of energy from oxidation of fatty acids, glucose, and glutamine were compared in cultures of chick embryo heart muscle (heart) and HeLa cells. Aerobic energy production, as measured by oxygen utilization, was comparable in the two cell types. In media containing dialyzed sera, the rates of incorporation of fatty acids directly into lipids were similar in both cells and accounted for greater than 97% of fatty acid metabolism in HeLa cells. However, in heart cells only 45% ended in lipid, 42% in protein, and 13% was released as CO2; the latter two products probably reflect the oxidation of fatty acids to acetyl-coenzyme A (-CoA) and its subsequent metabolism in the citrate cycle. Increased serum concentration in the medium did not affect fatty acid metabolism in HeLa cultures, but resulted in greater oxidation by heart cells (greater than 100 times that by HeLa cells). The metabolisms of both glucose and glutamine were similar in heart and HeLa cells with greater than or equal to 60% of glucose carbon ending as medium lactate and only 3-5% converted to acetyl-CoA. About 25% of glutamine carbon ended as CO2 and increased utilizations with increasing serum concentrations was accountable in both cells by increased lactate from glucose and glutamate from glutamine. CO2 production (and energy) from glutamine was independent of glutamine concentration within a tenfold range of physiological concentrations. The yields of energy have been calculated. In 10% dialyzed calf serum, oxidation of glutamine carbon provided about half of the total energy in heart cells; glucose about 35-45%, with most coming from glycolysis; oxidation of fatty acid carbon provided only 5-10%. That greater than 90% of the aerobic energy comes from glutamine in both cells can account for the comparable rates of oxygen utilization. HeLa cells derived little or no energy from fatty acids.