AbstractCytoplasmic dynein is essential for intracellular transport. Despite extensive in vitro characterizations, how the dynein motors transport vesicles by processive steps in live cells remains unclear. To dissect the molecular mechanisms of dynein, we develop optical probes that enable long-term single-particle tracking in live cells with high spatiotemporal resolution. We find that the number of active dynein motors transporting cargo switches stochastically between one and five dynein motors during long-range transport in neuronal axons. Our very bright optical probes allow the observation of individual molecular steps. Strikingly, these measurements reveal that the dwell times between steps are controlled by two temperature-dependent rate constants in which two ATP molecules are hydrolyzed sequentially during each dynein step. Thus, our observations uncover a previously unknown chemomechanical cycle of dynein-mediated cargo transport in living cells.