A new radiolabeled metabolite was released into the extracellular fluid by normal human skin fibroblasts that were labeled with [5,6,8,9,11,12,14,15-3H] arachidonic acid. This product continued to accumulate during a 24 h incubation, and its formation was not saturated at arachidonic acid concentrations up to 15 mumol/L. The compound, identified as hexadecatrienoic acid, was not produced by Zellweger fibroblasts which are deficient in peroxisomal fatty acid beta-oxidation. By contrast, radiolabeled hexadecatrienoic acid was produced by mutant fibroblasts having other peroxisomal defects, including X-linked adrenoleukodystrophy, adult Refsum's disease, and rhizomelic chondrodysplasia punctata. This radiolabeled metabolite also was produced by mutant fibroblasts that cannot oxidize long-chain fatty acids in the mitochondria. These results indicate that hexadecatrienoic acid is synthesized from arachidonic acid by peroxisomal beta-oxidation. The absence of this pathway may account for some of the biochemical and functional abnormalities that occur in Zellweger's syndrome.