A rapid autoradiographic screening procedure has been developed for identifying Chinese hamster ovary cell mutants defective in the peroxisomal enzyme dihydroxyacetonephosphate (DHAP) acyltransferase. Ten mutants were found among 60,000 colonies grown from a stock of mutagen-treated cells, and 3 have been characterized with respect to their enzymology and phospholipid biosynthesis. All three contain 3% (or less) of the parental DHAP acyltransferase activity measured at pH 5.5, the optimum for the peroxisomal enzyme. When measured at pH 7.4, all three contained 70-85% of the wild-type activity, but it was sensitive to N-ethylmaleimide. Glycerol-3-phosphate acyltransferase activities were identical in mutant and parent strains. Two other peroxisomal enzymes, alkyl-DHAP synthase and particulate catalase, were also reduced by factors of 5-10 in all three mutants, suggesting that these strains are deficient in some aspect of peroxisome assembly, possibly like cells from patients with Zellweger syndrome. Short-term and long-term labeling with 32Pi revealed that these mutants are grossly deficient in the de novo synthesis and content of plasmalogens. In parental cells the plasmalogen form of phosphatidylethanolamine constitutes 7.1% of the total phospholipid, but it is reduced to 0.7% in the mutants. This decrease is accompanied by a compensatory increase in the diacyl form of phosphatidylethanolamine. The results presented here support the view that there are two DHAP acyltransferases in animal cells and that the peroxisome is essential for the biosynthesis of plasmalogens.