The role of acetyl-coenzyme A carboxylase (ACC) in regulating fatty acid oxidation was investigated in isolated fatty acid perfused working rat hearts. Overall fatty acid oxidation rates were determined by addition of 1.2 mM [3H]palmitate to the perfusate of hearts in which the endogenous triglyceride pool was prelabeled with [14C]palmitate. Rates of both exogenous and endogenous fatty acid oxidation were measured by simultaneous measurement of 3H2O and 14CO2 production, respectively. A second series of hearts were perfused under similar conditions except that [U-14C]glucose was present in the perfusate for measurement of glucose oxidation rates. Addition of dichloroacetate (DCA, 1 mM) to the perfusate resulted in a dramatic stimulation of glucose oxidation (a 411% increase), with a parallel decrease in fatty acid oxidation (from 305 +/- 51 to 206 +/- 40 nmol/g dry weight.min.unit work). DCA treatment increased the contribution of glucose oxidation to ATP production from 7.1 to 30.6%, while decreasing the contribution of overall fatty acid oxidation from 92.9 to 69.4%. Tissue levels of malonyl-CoA in hearts treated with DCA were higher compared to controls (14.0 +/- 0.6 and 10.0 +/- 0.7 nmol/g dry weight, respectively) and were negatively correlated (r = −0.85) with overall fatty acid oxidation rates. Acetyl-CoA levels were also significantly higher in DCA-treated hearts, and a positive correlation (r = 0.88) was seen between myocardial acetyl-CoA and malonyl-CoA levels. This suggests that DCA treatment increased the supply of acetyl-CoA for ACC. Western blots revealed the presence of both the 280-kDa (ACC-280) and the 265-kDa (ACC-265) isoforms of ACC in cardiac tissue, with a predominance of ACC-280. The activity of ACC extracted from hearts was similar in both groups when assayed under optimal conditions of acetyl-CoA and citrate. However, using affinity purified ACC, it was demonstrated that heart ACC (predominantly ACC-280) had a higher Km for acetyl-CoA than ACC isolated from white adipose tissue (predominantly ACC-265). We conclude that ACC is an important regulator of fatty acid oxidation in the heart and that acetyl-CoA supply is a key determinant of heart ACC-280 activity. As acetyl-CoA levels increase, ACC-280 is activated resulting in an increase in malonyl-CoA inhibition of fatty acid oxidation.