The AS-30D rat hepatoma cell line is characteristic of that class of rapidly growing tumors which exhibit high rates of aerobic glucose utilization and lactic acid production (Bustamante, E., Morris, H.P., and Pedersen, P.L., J. Biol. Chem., 256: 8699-8704, 1981). In this study, we have examined the coupling properties of the mitochondria in intact AS-30D hepatoma cells and the relative contributions of cytoplasmic (glycolytic) and mitochondrial compartments to total cellular ATP production in the presence of glucose and glutamine. All respiration in AS-30D cells was inhibited by inhibitors of mitochondrial electron transport, ruling out significant rates of respiration from other cellular components. Moreover, cellular respiration was found to be coupled to phosphorylation of ADP, as demonstrated by its inhibition by oligomycin and aurovertin, inhibitors of the mitochondrial ATP synthetase (F0F1-ATPase). When intact cells were supplied with glucose as the only added energy source, it was estimated that about 60% of the total cell ATP was derived from glycolysis and 40% from oxidative phosphorylation. Addition of physiological concentrations of glutamine in the presence of glucose had little effect on the relative contributions of glycolysis and oxidative phosphorylation to total cellular ATP production. In the absence of added glucose, glutamine alone could maintain the same ATP production rates by supporting mitochondrial oxidative phosphorylation. It is concluded that, in the AS-30D hepatoma cell line, glucose is the preferred energy source, with the larger portion of ATP production being supplied by glycolytic reactions. Although oxidative substrates such as glutamine can replace glucose in maintaining total cell ATP production, they do not appear to be the major fuel sources when hepatoma AS-30D cells are exposed to concentrations of substrates which occur in vivo.