Using a monoclonal antibody (P6C3) derived from mice immunized with partially delipidated rat myelin, we detected a single 57.5 kDa protein species (BT57.5) positioned between alpha- and beta-tubulin on nitrocellulose containing electrophoretically separated rat brain myelin proteins. The kinetics of incorporation of 14C-labeled BT57.5 and myelin basic protein into myelin was similar in rats injected intracranially with [14C]amino acids to radioactively label these proteins. Immunoblotting of the separated proteins of the myelinlike fraction of rat brain with the anti-BT57.5 monoclonal antibody occasionally revealed a faint band corresponding to BT57.5 but consistently showed a more prominent protein band that migrated slightly ahead of BT57.5. When the myelin and myelinlike fraction were isolated from rats injected with [14C] amino acids, labeling of the prominent anti-BT57.5 binding protein in the myelinlike fraction preceded that of BT57.5 in myelin, reaching a peak of labeling and then decreasing before BT57.5 reached its peak level. Thus, the prominent anti-BT57.5 protein in the myelinlike fraction might be a metabolic precursor of BT57.5 in myelin or the myelinlike fraction is simply turning over more rapidly and is distinct from the myelin fraction. BT57.5 was detectable in rat, mouse, guinea pig, bovine, and human CNS myelin but not rabbit and was detectable in the peripheral nervous system only in myelin of rats and humans. Finally, in extensively purified myelin, BT57.5 appeared to copurify with myelin basic protein, suggesting that BT57.5 is a constituent of myelin rather than an artifact arising during brain homogenization procedures.