The GenS3 and B11F7 monoclonal antibodies (MAbs) have been widely used for biochemical and immunocytochemical experiments on myelin-associated glycoprotein (MAG), a cell adhesion molecule mediating the interaction between myelinating glia and axons. We have mapped the epitopes to within several amino acids on Ig domain 2 (D2) (amino acids 167-77) and domain 4 (D4) (amino acids 375-388) for GenS3 and B11F7, respectively. Domain deletion and substitution mutants of the MAG cDNA were first used to map the epitopes to a given domain. In the cases of GenS3, insertion mutants were used to resolve the epitope to a small region of D2. For the B11F7 epitope, a novel technique combining PCR and in vitro transcription and translation was used to generate small C-terminal deletions and map the epitope to 13 amino acids. Then, inhibition by peptides corresponding to the GenS3 (ELRPELSWLGHE; amino acids 167-177) and B11F7 (QLELPAVTPEDDGE; amino acids 375-388) epitopes was used to confirm the position of the epitopes based on the mutant data. Interestingly, the GenS3 epitope maps to a region predicted to be sequestered within the hydrophobic core of D2. This is consistent with the inability of GenS3 to recognize the epitope in native MAG; GenS3 epitope recognition occurs only in denatured MAG, where the epitope is more accessible. With the definition of the GenS3 and B11F7 epitopes, these antibodies will be useful for further structure-function studies on MAG.