Biochemical and cellular properties of three immortalized Schwann cell lines expressing different levels of the myelin-associated glycoprotein (MAG) were compared. The S16 line generated by repetitive passaging was described previously and expresses a level of MAG comparable to that in adult sciatic nerve. The S42 line was generated independently by the same procedure, divides more slowly than the S16 line, and expresses an even higher level of MAG. The S16Y line arose spontaneously from a passage of the S16 cells, divides much more rapidly, and does not express MAG. The levels of MAG expression in the three lines are inversely related to their rates of proliferation, and MAG mRNA levels parallel the amounts of MAG. The S16 and S42 lines consist mainly of flat cells at low density and develop many processes at high density, whereas most of the S16Y cells are spindle-shaped, resembling primary Schwann cells in appearance. Surface immunostaining with the O4 antibody was positive for the S16 and S42 cells and negative for the S16Y cells, but all three lines were negative for surface staining with the O1 antibody. The overall protein compositions of the three lines are very similar, but the S16 and S42 cells express larger amounts of several glycoproteins than the S16Y cells, including the adhesion proteins, neural cell adhesion molecule, L1, and laminin. S16 and S42 cells (but not S16Y cells) also express P0 glycoprotein, galactocerebroside, and sulfatide, but, unlike MAG, these other myelin-related components were present at much lower levels than in adult nerve. Myelin basic protein and proteolipid protein were not detected in any of the lines, although all three lines contained proteolipid protein mRNA. 2',3'-Cyclic nucleotide 3'-phosphodiesterase and glial fibrillary acidic protein were present in all three lines. Conditions have not yet been found in which any of the lines will myelinate dorsal root ganglion neurons in vitro, but the S16 and S42 cells differ from the S16Y cells by clustering around neurons after 1 week in coculture. In many respects, the S16 and S42 cells biochemically resemble Schwann cells at an early stage in their preparation to myelinate and should be useful for investigating the cell biology of MAG and other myelin-related components.