To screen fibroblasts for defects in lactate/pyruvate oxidation, cells were grown to confluence in (25-\mathrm{cm}^{2}) flasks, rinsed, and incubated in glucose-free media containing (25 \mu \mathrm{M}) L-lactate and (0.1 \mu \mathrm{Ci}\left[\mathrm{D}, \mathrm{L}-1-{ }^{14} \mathrm{C}\right]) lactate. Lactate oxidation was measured as the amount of lactate oxidized in nmol of ({ }^{14} \mathrm{CO}_{2}) generated /mg protein/ min. Fibroblasts from patients with mitochondrial or peroxisomal disorders had decreased lactate oxidation compared to the control (CON): CON, (1.9 \pm 0.13 \mathrm{nmol} /) (\mathrm{mg} / \mathrm{min}); neonatal adrenoleukodystrophy (NALD), 0.45 (\pm 0.01(P<0.001)); rhizomelic chondrodysplasia punctata (RCDP), (0.13 \pm 0.002(P<0.001)); mitochondrial defect of unknown etiology (MIT), (0.77 \pm 0.003(P) <0.001); pyruvate dehydrogenase (PDH) deficiency, (0.98 \pm 0.02(P<0.001)). This method is useful for screening fibroblasts for defects in lactate oxidation in patients with mitochondrial or peroxisomal disorders. Confirmation of the site of the defect may then be investigated with specific assays, e.g., PDH, in cellular homogenates: CON, (0.93 \pm 0.02 \mathrm{nmol} / \mathrm{mg} / \mathrm{min}); NALD, (0.55 \pm 0.02 ; R C D P, 0.44 \pm 0.02 ;) MIT, (0.53 \pm 0.03 ;) PDH deficiency, (0.19 \pm 0.02). (c) 1983 Academic Press, Inc.