We report that calf brain microtubules prepared wlthout nucleotide contain, In addition to kinesin and dynein, a polypeptide of 100 kd that could be dissociated by nucleotide. The protein was selectively extracted from microtubulea using a combination of GTP and AYPPNP. The extract contained mlcrotubulestimulated (g-fold) MgATPase activity that partitioned into two components upon further purification: the 100 kd polypeptide and a soluble activating fraction. The 100 kd protein induced mlcrotubules to form hexagonally packed bundles containing periodic cross bridges spaced 13 nm apart. In the presence of ATP and the activating fraction, bundles fragmented, elongated, and exhibited other behavior indlcatlve of sliding between microtubules. These findings indicate that the 100 kd protein is part of a novel mechanochemical enzyme, which we term Vynamlti, that may mediate microtubule sliding in viva.