The stepwise movements generated by myosin, dynein, and kinesin were observed in living cells in an attempt to understand the molecular mechanisms of movement within cells. First, the sequential process of the transport of vesicles, including human epidermal factor 2 receptor, after endocytosis was observed for long periods in three dimensions using quantum dots (QDs) and a three-dimensional confocal microscope. QD vesicles, after being endocytosed into the cells, moved along the membrane by transferring actin filaments and were then rapidly transported toward the nucleus along microtubules. Second, the position of vesicles was detected with a precision up to 1.9 nm and 330 micros using a new two-dimensional tracking method. The movement of the QDs transported by myosin VI lying just beneath the cell membrane consisted of 29- and 15-nm steps with a transition phase between these two steps. QD vesicles were then transported toward the nucleus or away from the nucleus toward the cell membrane with successive 8-nm steps. The stepwise movements of these motor proteins in cells were observed using new imaging methods that allowed the molecular mechanisms underlying traffic to and from the membrane to be determined.