Microtubule protein prepared from bovine brain by a temperature-dependent assembly-disassembly procedure contained Mg2+- or CA2+-stimulated ATPase activity. However, activity decreased with successive cycles of assembly-disassembly such tht 15% of the Mg2+-stimulated and 31% of the Ca2+-stimulated activity of the second-cycle material remained after seven cycles. Microtubule preparations purified by three cycles of assembly-disassembly contained many membrane fragments and vesicles which were absent in microtubule preparations cycled eight times. Histochemistry and electron microscopy revealed that much of the activity is associated with the vesicles. Vesicles with an accumulation of led phosphate deposits (indication of ATPase activity) were observed in high-speed pellets (150,000 g, 60 min) of microtubule-associated proteins. Most of the activity in the microtubule-associated protein preparations, but only a fraction of the total protein is pelleted. 53-78% of the ATPase activity, but only 6% of the total protein, is recovered in a microtubule-associated protein fraction eluted from phosphocellulose with 0.17 M NaCl. Polypeptides resolved on SDS polyacrylamide gradient gels have estimated molecular weights of 30,000-76,000. Electron micrographs of this material revealed short filaments, vesicles, and small ring-like structures. None of the inhibitors of possible contaminating ATPases affected the ATPase activity.