The initial steps of ether phospholipid biosynthesis take place in peroxisomes. Alkyl-dihydroxyacetonephosphate synthase, the peroxisomal enzyme that actually introduces the ether linkage, has been purified from guinea pig liver in this laboratory. With the amino acid sequences obtained from this protein, the authors were able to clone the cDNAs encoding this enzyme from both guinea pig and human liver. In both cases, the enzyme appears to be synthesized as a precursor protein with a N-terminal cleavable presequence containing a peroxisomal targeting signal (PTS) type 2. Levels of the enzyme protein were found to be strongly reduced in human fibroblasts derived from Zellweger syndrome and rhizomelic chondrodysplasia punctata patients. The molecular basis of an isolated alkyl-dihydroxyacetonephosphate synthase deficiency was resolved. A clone encoding a Caenorhabditis elegans homolog of the mammalian enzymes was characterized. In contrast to the mammalian enzymes, this C. elegans enzyme lacks a N-terminal PTS type 2 motif, but carries a C-terminal PTS type 1.