Turnover rate of individual molecular species of sphingomyelin of adult rat brain myelin and microsomal membranes was determined after an intracerebral injection of 100 microCi of [C3H3]choline. Myelin and microsomal membrane sphingomyelins were isolated from the rest of the lipids. The individual molecular species of benzoylated sphingomyelin were separated and quantitated by reversed-phase high performance liquid chromatography. All individual major molecular species of microsomal and myelin sphingomyelin had maximum incorporation at 6 and 15 days, respectively, after the injection. The specific radioactivity of all the various molecular species of both myelin and microsomal sphingomyelin declined at a similar rate after reaching a maximum. There was no significant difference in the turnover rate of short chain (16:0, 18:0) and long chain (greater than 22:0) fatty acid containing sphingomyelin. The average apparent turnover rate of myelin and microsomal sphingomyelin molecular species was about 14-16 days for the fast pool and about 45 days for the slow pool. It is concluded that individual molecular species of sphingomyelin of myelin and microsomal membranes turned over at a similar rate. Thus, turnover rate of sphingomyelin in myelin and microsomal membranes is not affected by the fatty acyl composition of the lipid.