Phosphorylation-dependent regulation of microtubule-stabilizing activities of microtubule-associated protein 2 (MAP2) was examined using optical microscopy. MAP2, purified from mammalian brain, was phosphorylated by either cAMP-dependent protein kinase (PKA) or cyclin B-dependent cdc2 kinase. Using PKA, 15 mol of phosphoryl groups was incorporated per mole of MAP2, but about 70% of the phosphates was distributed to the projection region. Using cdc2 kinase, 7-10 mol of phosphoryl groups was incorporated per mole of MAP2, and more than 60% of the phosphates was distributed to the microtubule-binding region. Both types of phosphorylation similarly reduced binding activity of MAP2 onto microtubules. Direct observation of individual microtubules using dark-field microscopy showed that interconversion between the polymerization phase and the depolymerization phase was repeated in both unphosphorylated and PKA-phosphorylated MAP2. In cdc2 kinase-phosphorylated MAP2, however, the phase transition from depolymerization to polymerization occurred with difficulty, with the result being that the half-life of individual microtubules was as short as in the absence of MAP2. Examination of spontaneous polymerization of microtubules using dark-field microscopy showed that the microtubule-nucleating activity of MAP2 was reduced by PKA-dependent phosphorylation and was completely abolished by cdc2 kinase-dependent phosphorylation. These observations show that cdc2 kinase-dependent phosphorylation inhibits both the microtubule-stabilizing activity and the microtubule-nucleating activity of MAP2, while PKA-dependent phosphorylation affects only the microtubule-nucleating activity of MAP2.