The microtubule-binding domains of microtubule-associated protein (MAP) 2, MAP4, and tau are structurally similar [Aizawa, H., Emori, Y., Murofushi, H., Kawasaki, H., Sakai., H., and Suzuki, K. (1990) J. Biol. Chem. 265, 13849-13855]. To compare the microtubule-binding mechanisms of the three MAPs, we performed a quantitative competition analysis using the three MAPs and the microtubule-binding domain fragment of MAP4 (PA4T fragment). The two-cycled microtubule protein fraction from bovine brain contains MAP1, MAP2, MAP4, and tau. When an excess of the PA4T fragment was added to the microtubule protein fraction, MAP4 and tau were completely released from the microtubules, while MAP1 remained bound. MAP2 was only partially released from the microtubules. The competition between MAP2 and MAP4 was further analyzed using purified MAP2, the PA4T fragment, and tubulin. About half of the MAP2 was still bound to the microtubules, even in the presence of an excess amount of the PA4T fragment. The microtubule-binding mechanisms of MAP2 and MAP4 seem to be different, in spite of their similar primary structures.