Recombinant DNA methods were used to obtain soluble, undenatured fragments of the heavy chain of myosin subfragment 1 (S-1). These fragments were of preselected lengths and could include protease-sensitive segments that are destroyed when other preparation methods are used. Actin binding by each of the three contiguous segments (residues 1-248, 249-524, and 518-722, essentially spanning the entire S-1 heavy chain) was demonstrated. ATP binding, comparable to that of native S-1, was obtained only with a segment consisting of residues 1-524. Competition among the various fragments for actin was also studied. The data are discussed in relation to the recently reported resolved structure of S-1 [Rayment, I., Rypnieski, R. W., Schmidt-Bäse, K., Smith, R., Tomchick, D. R., Benning, M. M., Winkelmann, D. A., Wesenberg, G. & Holden, H. M. (1993) Science 261, 50-58].