Myelin-associated glycoprotein (MAG) has been implicated in the mediation of interactions between oligodendrocytes and neurons during the development of the myelin sheath. Here we show that MAG is phosphorylated in intact myelinating mouse brain primarily at serine residues and to a lesser extent at threonine and tyrosine residues. In vivo, only the larger of the two developmentally regulated MAG isoforms is phosphorylated. MAG can be phosphorylated at tyrosine by the v-fps and v-src protein-tyrosine kinases in vitro and by a kinase endogenous to myelin membrane preparations. MAG phosphorylated in myelin membranes in vitro also contains phosphoserine and phosphothreonine. These observations suggest that phosphorylation of MAG is physiologically significant in regulating oligodendrocyte-neuron interactions.