The α-oxidation of phytanic acid and the β-oxidation of pristanitc acid were investigated in cultured fibroblasts from controls and patients affected with different peroxisomal disorders using deuterated substrates. Formation of [ω-2H6]4,8-dimethylnonanoylcarnitine ([ω-2H6]C11-carnitine) from [ω-2 H6]phytanic acid and [ω-2H6]pristanic acid was used as marker for these processes. Analysis was performed by tandem mass spectrometry. In normal cells, formation of [ω-2H6]C11-carnitine from both [ω-2H6]phytanic acid and [ω-2H 6]pristanic acid was observed. When peroxisome-deficient fibroblasts were incubated with these substrates, [ω-2H6]C11-carnitine was not detectable or, in two cases, very low, which results from deficiencies in both peroxisomal α- and β-oxidation. In cells with an isolated β-oxidation defect at the level of the peroxisomal bifunctional protein, formation of [ω-2H6]C11-carnitine could also not be detected. Cells with an isolated defect in the α-oxidation of phytanic acid, obtained from patients affected with Refsum disease (McKusick 266500) or rhizomelic chondrodysplasia punctata (McKusick 215100), did not form [ω-2H6]C11 -carnitine from [ω-2H6]phytanic acid. The observed formation of [ω-2H6]C11-carnitine from [ω-2H6]pristanic acid in these cells is in accordance with a normal peroxisomal β -oxidation in these disorders. This study shows that separate incubation of fibroblasts with [ω-2H 6]phytanic acid and [ω-2H6]pristanic acid, followed by acylcarnitine analysis in the medium by tandem mass spectrometry, can be used for screening cell lines for deficiencies in the peroxisomal α- and β-oxidation pathways.