Previous reports from several laboratories have demonstrated the presence of many lipid-metabolizing enzymes in myelin, including all the enzymes needed to convert diacylglycerol to phosphatidylcholine and phosphatidylethanolamine. Axonal transport studies had suggested the presence of additional enzymes which incorporate acyl chains into specific phospholipids of myelin. We report here evidence for one such group of enzymes, the acyl-CoA:lysophospholipid acyltransferases. At the same time, activity of acyl-CoA:sn-glycerol-3-phosphate acyltransferase was negligible in myelin. Oleoyl-CoA and arachidonoyl-CoA were both active substrates for transfer of acyl chains to lysophosphatidylcholine and lysophosphatidylinositol. Activity in myelin varied from 7 to 19% of microsomal activity, values well above the likely level of microsomal contamination as judged by microsomal markers. Additional evidence for a myelin locus came from assays at sequential stages of purification and from mixing experiments. Arachidonoyl-CoA was somewhat more reactive than oleoyl-CoA toward lysophosphatidylcholine; the myelin Km for these two CoA derivatives was 98 microM and 6.6 microM, respectively. Activity with lysophosphatidylinositol as substrate was approximately 40% of that with lysophosphatidylcholine in myelin, whereas activities with lysophosphatidylethanolamine and lysophosphatidylserine were considerably less.