Dynein is a motor ATPase, and the C-terminal two-thirds of its heavy chain form a ring structure. One of protrudings from this ring structure is a stalk whose tip, the dynein stalk head (DSH), is thought to be the microtubule-binding domain. As a first step toward elucidating the functional mechanisms of DSH, we aimed at the NMR structural analysis of an isolated DSH from mouse cytoplasmic dynein. The DSH expressed in bacteria and purified was coprecipitated with microtubules, suggesting its proper folding. Chemical shifts of the DSH were obtained from NMR measurements, and backbone assignment identified 94% of the main-chain N–H signals. Secondary structural prediction programs showed that about 60% of the residues formed α-helices. A region with cationic residues K58 and R61 (and possibly R66 as well), and another with R86, K88, K90, and K91, were found to form α-helices. Both of these regions may be important in the formation of the DSH-binding site to a microtubule that has a low pI with a number of acidic residues. Two synthetic peptides containing the sequence of the α-helix 12 of β-tubulin, considered to be important in binding to DSH, were investigated. Of these two peptides, the one with higher helix-formation propensity appeared to bind to DSH, since it precipitated with DSH in a nearly stoichiometric manner. This suggested that the α-helicity of this region would be important in its binding to DSH.