Myelin basic protein has been used as a model substrate for determination of substrate recognition motif of various protein kinases. In this report phosphorylated sites of bovine brain myelin basic protein were studied with a catalytic fragment of protein-tyrosine kinase p72syk. Three of four tyrosine residues in myelin basic protein were phosphorylated by this kinase. Major phosphorylated site was 134Y and minor phosphorylated sites were 68Y and 127Y. As the phosphorylation site by p56lck was only 68Y, the recognition motif of p72syk was quite different from that of p56lck. Furthermore, the fact that elution pattern on HPLC of the phosphopeptides obtained by insulin receptor kinase was different from that by p72syk suggested that the recognition motif of p72syk was also different from that of insulin receptor kinase. These results may suggest that each protein-tyrosine kinase has a specific substrate recognition motif.