Inherited defects in myocardial long-chain fatty acid metabolism are increasingly recognized as a cause of cardiomyopathy and sudden death in children. To evaluate whether the phenotypic expression of these genetic diseases could be delineated using positron emission tomography (PET), 11 patients with inherited defects in fatty acid metabolism were evaluated and results were compared with those of 6 nonaffected siblings. Myocardial perfusion, myocardial oxygen consumption (MVO₂), and long-chain fatty acid metabolism were determined noninvasively with PET using quantitative mathematical models. There were no differences in haemodynamics, perfusion, MVO₂ or plasma substrate levels between groups. Patients with defects in enzymes of fatty acid β-oxidation (acyl-CoA dehydrogenase and 3-hydroxyacyl-CoA dehydrogenase deficiencies) n = 5 had diminished myocardial palmitate oxidation compared with healthy siblings (3.2 ± 3.0 vs 13.0 ± 5.6 nmol/g per min, p < 0.03) and a decrease in the percentage of MVO₂ accounted for by palmitate (2% ± 3% vs 9% ± 5%), p > 0.04). In these patients, extracted palmitate was shunted into a slow-turnover compartment (predominantly reflecting esterification to triglycerides) with expansion of palmitate in that pool (185 ± 246 compared with 27 ± 67 nmol/g in healthy siblings, p < 0.02). In contrast, myocardium of patients with carnitine deficiency (n = 6 (all on oral carnitine therapy) had normal palmitate extraction but expansion of the interstitial/cytosolic fatty acid pool (617 ± 399 vs 261 ± 73 nmol/g in healthy siblings, p < 0.04), suggesting different mechanisms for handling upstream fatty acyl intermediates. Thus, PET can be used to noninvasively assess abnormal myocardial handling of fatty acids in patients with inherited defects of metabolism. This approach should be useful in the assessment of altered myocardial fatty acid metabolism associated with cardiomyopathy as well as for evaluating the efficacy of therapeutic interventions in affected patients.