Most studies on the pathological responses of the heart to ethanol have been conducted in isolated systems. The objectives of this study were to determine (1) the synthesis rate of ventricular mitochondrial proteins in vivo and (2) whether the synthesis rates of these proteins are perturbed by acute ethanol exposure in vivo. Fractional rates of protein synthesis [defined as the percentage of tissue protein renewed each day; i.e., ks (%/day)] were determined in male Wistar rats by in vivo injection of a flooding dose of L-[4-3H] phenylalanine. Subsarcolemmal mitochondria were released by polytron treatment, and the isolation of interfibrillar mitochondria involved treatment of the cardiac homogenate with the proteolytic enzyme Nagarse. In the control rats mean ks values of 22.4%/day were observed for mixed cardiac proteins. The synthesis rates of subsarcolemmal and interfibrillar mitochondrial proteins were lower, i.e., 16.9%/day and 10.9%/day, respectively. Acute ethanol administration (75 mmol/kg body weight ip, 2.5 hr) depressed the fractional rate of protein synthesis in all cardiac fractions, including those pertaining to the mitochondria, as follows: mixed fraction--21%, p < 0.01; subsarcolemmal mitochondria--23%, p < 0.01; interfibrillar mitochondria--26%, p < 0.05; and nuclear fraction--20%, p < 0.05. In conclusion, the reduced synthesis rate of the mitochondrial proteins in response to acute ethanol exposure may in some way be partly connected with the depression in myocardial contractility and associated functional damage of mitochondrial metabolism.