We have previously reported that the peroxisomal beta-oxidation system for very long chain fatty acids is defective in X-linked childhood adrenoleukodystrophy [(1984) Proc. Natl. Acad. Sci. USA 81, 4203-4207]. In order to elucidate the specific enzyme defect, we examined the oxidation of [1-14C]lignoceric acid, [1-14C]lignoceroyl-CoA and (1-14C)-labelled alpha,beta-unsaturated lignoceroyl-CoA (substrates for the 1st, 2nd, and 3rd steps of the beta-oxidation cycle, respectively). These studies suggest that the pathognomonic accumulation of very long chain fatty acids in X-linked childhood ALD may be due to the defective activity of peroxisomal very long chain (lignoceroyl-CoA) acyl-CoA ligase.