Although the myelin-associated glycoprotein (MAG) cannot be detected in primary cultures of rat Schwann cells in the absence of neurons, MAG expression was demonstrated in some lines of cultured Schwann cells that had been immortalized by repetitive passaging. Radioimmunoassay of one such Schwann cell line, S-16, showed a remarkably high MAG concentration of about 1 ng/microgram of total protein, a level that is comparable to the MAG concentration in adult sciatic nerve. The S-16 cells divide very rapidly, are rounder than normal Schwann cells, and elaborate many processes after reaching high density. The cells are galactocerebroside positive, but express little or no P0 glycoprotein or myelin basic protein. As in nerve, the MAG synthesized by the cultured cells is primarily the shorter isoform (S-MAG). Furthermore, the posttranslational processing resembles that occurring in vivo including a similar degree of glycosylation, sulfation of oligosaccharides, and phosphorylation of the polypeptide. The sensitivity of MAG to treatment of the intact cells with trypsin or neuraminidase, as well as surface labeling with [3H]borohydride reduction after periodate oxidation, demonstrated that most of the MAG expressed by the S-16 cells is located on the cell surface. This line of immortalized Schwann cells expressing a remarkably high level of MAG should be useful for investigating the cell biology and function of this glycoprotein.