Effects of fructose and glucose on cardiac function and 14CO2 production from radioactive hexoses were studied in isolated working guinea pig hearts perfused at constant filling pressure via the left atrium. When hearts were perfused without exogenous substrates external work declined, indicating exhaustion of endogenous energy sources. It was possible to restore left ventricular function with concentrations of glucose in the physiological range; in order to achieve a similar restoration with fructose as the only exogenous substrate concentrations higher than 40mM were necessary. 14CO2 necessary. 14CO2 production from [U-14C]glucose was half-maximal at about 2mM glucose. On the other hand, 14CO2 production from [U-14C]fructose did not show saturation kinetics and, at concentrations around 2mM, was less than 5% of the rates observed with [U-14C]glucose. Moreover, fructose oxidation was suppressed in the presence of 5mM glucose. The data suggest that fructose cannot serve as a major substrate in the heart under in vivo conditions.