The terminal step during aerobic plasmalogen biosynthesis is catalyzed by a microsomal desaturase system which converts 1-O-alkyl-2-acyl-sn-glycerophosphoethanolamine to 1-O-alk-1'-enyl-2-acyl-sn-glycerophosphoethanolamine (ethanolamine plasmalogen). The reaction depends on oxygen and NAD(P)H and is stimulated 3-10-fold by soluble activating factors contained in the 100 000 X g supernatant. Two stimulating proteins have been isolated from pig kidney; the partially purified proteins have identical molecular weights (27 000) but differ in their respective isoelectric points (protein I, 5.1 and protein II, 4.9). Both proteins behave identically in the biochemical studies conducted. Exogenous substrate binds to the stimulating proteins; the transfer of ethanolamine, but not of choline phospholipids, from liposomes to microsomes is enhanced by the stimulating proteins. They stimulate plasmalogen synthesis from either exogenous or endogenous substrate (synthesized from alkylglycerophosphoethanolamine by microsomal transacylases). The stimulating proteins have no enzymatic activity themselves; it is suggested that they affect events within the membrane and function as specific mediators between the membrane-bound enzyme system and the lipophilic substrate.