Dietary management of long-chain l-3-hydroxyacyl CoA dehydrogenase (LCHAD) deficiency involves a regimen that contains adequate carbohydrate, protein, and essential lipids, and supplementation with medium-chain fatty acids in the form of medium-chain triglycerides, providing energy from fats that bypasses the long-chain metabolic block. This study analyzes the effects of dietary treatment of LCHAD deficiency in an in vitro model. Cultured skin fibroblasts from LCHAD-deficient and normal individuals were grown in media supplemented with physiologic combinations of medium-chain fatty acids octanoate and decanoate, and the long-chain palmitate. Medium was removed from the cells after various incubation times, and assayed for 3-hydroxy-intermediates of fatty acid oxidation. The 3-hydroxy-fatty acids were measured by stable-isotope dilution gas chromatography/mass spectrometry. We found that the addition of medium-chain fatty acids caused a decrease in the accumulation of long-chain fatty acid oxidation intermediates in LCHAD-deficient cells when the cells were incubated in untreated medium, and also when they were incubated in this medium with palmitate added. Medium with decanoate alone was better at achieving this effect than medium with only octanoate added. A 1:3 ratio of octanoate to decanoate worked best over an extended time period in LCHAD-deficient cells in untreated medium, whereas a 1:1 ratio of octanoate to decanoate worked best in the same cells incubated in medium containing palmitate. In all dietary medium-chain triglyceride preparations, the ratio of octanoate was greater than that of decanoate. Our results suggest that a medium-chain triglyceride preparation that is higher in decanoate may be more effective in reducing the accumulation of potentially toxic long-chain 3-hydroxy-fatty acids in LCHAD deficiency.