The sequence of reactions involved in plasmalogen biosynthesis has been evaluated in cultured fibroblasts of patients with the cerebrohepatorenal syndrome. A double-label, double-substrate incubation using [1-14C] hexadecanol and 1-0-[9', 10'-3H]hexadecylglycerol was performed to monitor the relative rates of peroxisomal and microsomal biosynthesic steps. [14C] radioactivity associated with 1'-alkenyl groups of plasmalogens was found to be drastically reduced in fibroblasts of affected patients whereas [3H] incorporation was apparently normal. This finding is specific for cerebrohepatorenal syndrome fibroblasts since cell lines of patients with childhood adrenoleukodystrophy and neuronal ceroidlipofuscinosis utilized the lipid precursors of plasmalogen biosynthesis at normal rates. The results show that the defect in plasmalogen synthesis in the cerebro-hepato-renal syndrome is restricted to the peroxisomal steps. The finding of normal microsomal biosynthetic steps was exploited to devise a novel diagnostic assay in fibroblasts and amniocytes based on the comparison of [3H/14C] isotope ratios within aldehydes released from plasmalogens by acid hydrolysis. The procedure can be completed with a minimal amount of cells since it renders quantitative analyses unnecessary. Therefore, this technique appears ideally suited for the sensitive and safe prenatal diagnosis of the cerebro-hepato-renal syndrome.