The study of cellular processes such as organelle transport often demands particle tracking with microsecond time-resolution and nanometer spatial precision, posing significant challenges to existing tracking methods. Here, we have developed a novel strategy for two-dimensional tracking of gold nanoparticles (GNPs) with 25 mus time resolution and approximately 1.5 nm spatial precision, by using a quadrant photodiode to record the positions of GNPs in an objective-type dark-field microscope. In combination with a feedback loop, this technique records long, high time-resolution and spatial precision trajectories of endocytosed GNPs transported by the molecular motors kinesin and dynein in a living cell. In the full range of organelle velocities (0-8 microm s(-1)), we clearly resolve the individual 8 nm steps of cargoes carried by kinesin, and the 8, 12, 16, 20, and 24 nm steps of those carried by dynein. These experiments yield new information about molecular motor stepping in living cells.