Using the CHO2 monoclonal antibody raised against CHO spindles (Sellitto, C., M. Kimble, and R. Kuriyama. 1992. Cell Motil. Cytoskeleton. 22:7-24) we identified a 66-kD protein located at the interphase centrosome and mitotic spindle. Isolated cDNAs for the antigen encode a 622-amino acid polypeptide. Sequence analysis revealed the presence of 340-amino acid residues in the COOH terminus, which is homologous to the motor domain conserved among other members of the kinesin superfamily. The protein is composed of a central alpha-helical portion with globular domains at both NH2 and COOH termini, and the epitope to the monoclonal antibody resides in the central alpha-helical stalk. A series of deletion constructs were created for in vitro analysis of microtubule interactions. While the microtubule binding and bundling activities require both the presence of the COOH terminus and the alpha-helical domain, the NH2-terminal half of the antigen lacked the ability to interact with microtubules. The full-length as well as deleted proteins consisting of the COOH-terminal motor and the central alpha-helical stalk supported microtubule gliding, with velocity ranging from 1.0 to 8.4 mu m/minute. The speed of microtubule movement decreased with decreasing lengths of the central stalk attached to the COOH-terminal motor. The microtubules moved with their plus end leading, indicating that the antigen is a minus end-directed motor. The CHO2 sequence shows 86% identify to HSET, a gene located at the centromeric end of the human MHC region in chromosome 6 (Ando, A., Y. Y. Kikuti, H. Kawata, N. Okamoto, T. Imai, T. Eki, K. Yokoyama, E. Soeda, T. Ikemura, K. Abe, and H. Inoko. 1994. Immunogenetics. 39:194-200), indicating that HSET might represent a human homologue of the CHO2 antigen.