Identification of the ATPase involved in fast axonal transport of membranous organelles has proven difficult. Myosin and dynein, other ATPases known to be involved in cell motility, have properties that are inconsistent with the established properties of fast axonal transport, an essential component of which is readily solubilized in physiological buffer conditions rather than being stably associated with either membranous organelles or cytoskeletal elements. Adenylyl imidodiphosphate (AMP-PNP), a nonhydrolysable analogue of ATP, is a potent inhibitor of fast axonal transport that results in a stable interaction of membranous organelles with microtubules. Here we report the identification and partial characterization of an ATPase activity from brain whose binding to microtubules is stabilized by AMP-PNP. This ATPase activity seems to be associated with a polypeptide of relative molecular mass (Mr) 130,000 that is highly enriched in microtubule pellets after incubation with AMP-PNP and a soluble fraction from chick brain. This novel ATPase fraction has the predicted characteristics of the motor involved in fast axonal transport. Common features between the ATPase and fast axonal transport include interaction with the cytoskeleton in the presence of AMP-PNP, ready extractability, no Ca2+ dependence and inhibition by EDTA.