The Zellweger cerebro-hepato-renal syndrome is a genetic disease characterized by the absence of peroxisomes and deficiency of glycerol-ether lipids in several tissues. We measured the activity of dihydroxyacetone phosphate (DHAP) acyltransferase, a peroxisomal enzyme with a major role in ether lipid synthesis, in fibroblasts and leukocytes from patients with Zellweger syndrome. Control skin and amniotic-fluid fibroblasts had normal activity of DHAP acyltransferase (0.28 to 0.3 nmol per minute per milligram of protein), whereas fibroblasts from three patients with Zellweger syndrome had deficient activity (0.013 +/- 0.006 nmol per minute per milligram of protein). The activity of the enzyme in leukocytes and levels of plasmalogens (the major class of cellular glycerol-ether lipids) in erythrocytes were also deficient in a patient, but normal levels of leukocyte enzyme and erythrocyte plasmalogens were found in her parents. Other enzymes of the acyl DHAP pathway exhibited alterations in fibroblasts from patients with Zellweger syndrome, and the activity of the glycerophosphate acyltransferase was also reduced. These results support prior studies emphasizing the role of peroxisomes and the acyl DHAP pathway in cellular ether lipid synthesis, establish Zellweger syndrome cells as valuable for elucidating peroxisomal functions, and provide prenatal and postnatal diagnostic assays as well as potential therapeutic strategies for Zellweger syndrome.