Alkyldihydroxyacetone-P (alkyl-DHAP) synthase, the enzyme that forms the ether linkage of alkyl and alk-1-enyl glycerolipids, has been solubilized from ehrlich ascites cell microsomes using Triton X-100 and acetone precipitation. the solubilized enzyme, which is stabilized by glycerol or ethylene glycol, was then purified further by chromatography on DEAE-cellulose, QAE-Sephadex, Matrex Red, and hydroxylapatite with the aid of a new rapid assay method using DEAE-cellulose disks. Four enzymes involved in the metabolism of acyl-DHAP and alkyl-DHAP (acyl-DHAP/alkyl-DHAP oxidoreductase, DHAP acyltransferase, alkyl-DHAP phosphohydrolase, and a dinitrofluorobenzene-insensitive acyl-DHAP acylhydrolase) are removed under these conditions along with endogenous fatty acids and fatty alcohols. Two other activities copurify with the alkyl-DHAP synthase forward reaction: an acyl exchange reaction, in which [1-14C]palmitic acid is incorporated into palmitoyl-DHAP, and an alkyl exchange reaction, in which [1-14C]hexadecanol is incorporated into hexadecyl-DHAP. Exchange reactions of this type are characteristic properties of a ping-pong mechanism but not a sequential mechanism. This is confirmed by documentation that palmitic acid is a competitive inhibitor with respect to hexadecanol. In addition, low levels of palmitoyl-DHAP (less than 100 microM) show competitive inhibition with respect to hexadecanol, possibly due to palmitic acid formed from palmitoyl-DHAP by alkyl-DHAP synthase under these conditions. Based on the observations presented here and previously, a molecular mechanism for alkyl-DHAP synthase is proposed.