The effects of aspirin metabolites on beta-oxidation were studied in skin fibroblasts from eight typical Reye's syndrome (RS) patients and controls. RS patients' cells did not differ from controls in rates of palmitate oxidation or in the three component activities of the mitochondrial trifunctional enzyme (MTE), indicating no inherited beta-oxidation defect. Aspirin metabolites salicylate, hydroxyhippurate and gentisate, but not aspirin, directly inhibited palmitate oxidation in control and RS cells. RS cells were significantly more sensitive to inhibition than controls at 0.5 to 5 mM salicylate. Inhibition was concentration-dependent and reversible. Inhibition did not occur in fibroblasts lacking activity of the long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) activity of MTE. Salicylate was therefore inhibiting beta-oxidation at this step. Hydroxyhippurate and salicylate reversibly inhibited HAD activities in extracts of control and RS cells. Studies with pure short-chain HAD and LCHAD (MTE) showed hydroxyhippurate and salicylate were competitive inhibitors of the former but mixed (not competitive) inhibitors of the latter. Both compounds inhibited the combined, three-step, MTE reaction measured in the physiological direction. We conclude that (1) salicylate and hydroxyhippurate decrease beta-oxidation in intact cells by reversible inhibition of LCHAD activity of the MTE, and (2) beta-oxidation in RS cells is inherently more sensitive to inhibition by low concentrations of salicylate than controls.