Calcineurin dephosphorylated microtubule-associated protein 2 (MAP2) and tau factor phosphorylated by cyclic AMP-dependent and Ca2+, calmodulin-dependent protein kinases from the brain. Tubulin, only phosphorylated by the Ca2+, calmodulin-dependent protein kinase, served as substrate for calcineurin. The concentrations of calmodulin required to give half-maximal activation of calcineurin were 21 and 16 nM with MAP2 and tau factor as substrates, respectively. The Km and Vmax values were in ranges of 1-3 microM and 0.4-1.7 mumol/mg/min, respectively, for MAP2 and tau factor. The Km value for tubulin was in a similar range, but the Vmax value was lower. The peptide map analysis revealed that calcineurin dephosphorylated MAP2 and tau factor universally, but not in a site-specific manner. The autophosphorylated Ca2+, calmodulin-dependent protein kinase was not dephosphorylated by calcineurin. These results suggest that calcineurin plays an important role in the functions of microtubules via dephosphorylation.