Myelin-associated glycoprotein (MAG) is a cell surface molecule expressed by oligodendrocytes and Schwann cells. In order to determine whether MAG expression can confer adhesive properties to cells which normally do not aggregate in suspension, the cDNA encoding the long form of MAG (L-MAG) was introduced into L cell fibroblasts by retroviral infection. Clonal L cell lines expressing MAG were then subjected to a cell aggregation assay. Our results indicate that L-MAG can function as an intercellular adhesion molecule in a heterologous cell system. A critical threshold value of L-MAG expression was required for cell aggregation to occur. The adhesive properties of these cells were specific to MAG, since monoclonal antibodies directed against its extracellular domain inhibited aggregation. Furthermore, the adhesion was found to be calcium- and temperature-independent. Cell sorting experiments demonstrated that L-MAG-expressing cells bind in a heterotypic fashion to parental L cell fibroblasts. These results suggest that L-MAG can function as a heterotypic cell adhesion molecule recognizing a cell surface molecule(s) expressed by L cells.