Microtubule-associated protein 2 (MAP2) is an in vitro substrate for MAP kinase. Part of the phosphorylation occurs at the C-terminal microtubule-binding domain of the molecule which contains a cluster of putative consensus sites for MAP kinase on a proline-rich region. A peptide with the sequence RTPGTPG-TPSY, located at this region of the molecule, is efficiently phosphorylated by MAP kinase in vitro. An antibody (972) raised against this non-phosphorylated peptide has been used to test for in vivo phosphorylation at the proline-rich domain of the MAP2 molecule. The reaction of purified MAP2 with antibody 972 diminishes after in vitro phosphorylation by MAP kinase and is enhanced after in vitro dephosphorylation by alkaline phosphatase. A fraction of brain MAP2 isolated by iron-chelation affinity chromatography appears to be phosphorylated in vivo at the site recognized by antibody 972. There is some variation in the phosphorylation of MAP2 at the proline-rich region throughout rat brain development. MAP2C is more highly phosphorylated in the developing rat brain, whereas high-molecular-mass MAP2 is more extensively phosphorylated in the adult rat brain.