P ristanic acid oxidation measurements proved a reliable tool for assessing complementation in fused heterokaryons from patients with peroxisomal biogenesis defects. We, therefore, used this method to determine the complementation groups of patients with isolated defects in peroxisomal beta-oxidation. The rate of oxidation of pristanic acid was reduced in affected cell lines from all of the families with inherited defects in peroxisomal beta-oxidation, thus excluding the possibility of a defective acyl CoA oxidase. Complementation analyses indicated that all of the patients belonged to the same complementation group, which corresponded to cell lines with bifunctional protein defects. Phytanic acid oxidation was reduced in fibroblasts from some, but not all, of the patients. Plasma samples were still available from six of the patients. The ratio of pristanic acid to phytanic acid was elevated in all of these samples, as were the levels of saturated very long chain fatty acids (VLCFA). However, the levels of bile acid intermediates, polyenoic VLCFA, and docosahexaenoic acid were abnormal in only some of the samples. Pristanic acid oxidation measurements were helpful in a prenatal assessment for one of the families where previous experience had shown that cellular VLCFA levels were not consistently elevated in affected individuals.