Calmodulin binding to microtubule-associated proteins (MAPs) was studied by using three experimental techniques: affinity chromatography, cross-linking, and equilibrium binding. 1) Calmodulin affinity chromatography: both MAP2 and tau proteins were bound to calmodulin affinity columns in the presence of calcium and released with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), whereas tubulin was not bound. 2) Cross-linking 125I-calmodulin to whole MAPs and MAP2 by disuccinimidyl suberate: 125I-calmodulin was cross-linked to MAP2 and tau proteins showing an intense radioactivity band at 300,000 daltons and a diffuse band between 70,000 and 90,000 daltons. The cross-linking was calcium-dependent and was blocked by EGTA, trifluoperazine, or excess unlabeled calmodulin. 3) Equilibrium binding of 125I-calmodulin to MAP2 and tau using the Hummel-Dreyer technique on Sephadex G-100 columns: MAP2 and tau proteins bound 125I-calmodulin in a calcium-dependent manner and no binding occurred in the presence of EGTA. The apparent dissociation constant of calmodulin for MAP2 was 7 microM. The results indicate that calmodulin exerts its effect on microtubule assembly through the formation of a "Ca2+ X calmodulin X MAP2 or X tau" complex.