The application of radiolabeled fatty acids to measurements of fatty acid oxidation is discussed and a method for measuring the rate of beta-oxidation and of acetyl-CoA oxidation to CO2 is described. In hepatocytes from starved or fed rats, ethanol inhibited total beta-oxidation in the presence of 1.3 mM palmitate by 22% and 25%, respectively. If changes in the specific radioactivity of acetyl-CoA were not corrected for, the effect of ethanol would have been overestimated by 15% and underestimated by 15% in hepatocytes from fed and starved rats, respectively. In perfused liver from fed rats, inhibition by ethanol of total beta-oxidation in the presence of 1 mM palmitate was 35%. The rate of beta-oxidation in the absence of ethanol was underestimated by 65% if proper corrections were not applied. Inhibition of the tricarboxylic acid cycle by ethanol was 57% and 72% in hepatocytes from starved and fed rats, respectively. Pyrazole titration experiments demonstrated a correlation between changes in the mitochondrial NADH/NAD+ ratio and both inhibition of the tricarboxylic acid cycle and inhibition of the beta-oxidation pathway. The concentration of acetoacetyl-CoA is suggested as an additional regulatory factor of the beta-oxidation pathway. The ethanol-induced accumulation of triacylglycerol as a consequence of the inhibition of the beta-oxidation pathway is estimated to represent a 10% increase in the cellular triacylglycerol pool/hr/g of wet weight. Hence its chemical determination requires experiments of several hours duration. Primary cultures of hepatocytes have been shown to be a useful experimental system for studies of the ethanol-induced triacylglycerol accumulation.