The activities of 5'-nucleotidase, measured in brain homogenates and myelin isolated from rats at 21, 60 and greater than 90 days of age, were compared to values for two other myelin-associated enzymes, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) and carbonic anhydrase. Whereas the activities of all 3 enzymes were higher in brain homogenates from 60-day-old rats than in those from 21-day-old rats, only 5'-nucleotidase increased significantly in specific activity in both homogenates and myelin after the age of 60 days. The ratios of 5'-nucleotidase to the myelin basic and proteolipid proteins in subcellular fractions from adult rat brain suggested that the microsomal fraction was the only fraction containing 5'-nucleotidase levels not attributable to contamination by myelin membranes. Like carbonic anhydrase, 5'-nucleotidase had a greater distribution than CNP into microsomes of adult rats. When purified myelin was fractionated on a density gradient, the specific activity of 5'-nucleotidase was highest in the heaviest subfraction, with recovery of significant activity occurring, however, in all 3 subfractions. In rats over 60 days of age the recovery of 5'-nucleotidase in myelin was almost as high as that of the relatively myelin-specific enzyme CNP, suggesting that myelin may be the predominant, although not exclusive locus of 5'-nucleotidase in the adult rat brain.