We have developed a new technique for imaging single fluorescent dye molecules by refining epifluorescence and total internal reflection fluorescence microscopies. In contrast to previously reported single fluorescent molecule imaging methods, in which specimens were immobilized on an air-dried surface, our method enables video-rate imaging of single molecules in aqueous solution, This approach enabled us to directly image the processive movement of individual fluorescently labeled kinesin molecules along a microtubule. This method was also used to visualize individual ATP turnover reactions of single myosin molecules. The method can be combined with molecular manipulation using an optical trap. A single kinesin molecule attached to a polystyrene bead was brought into contact with a microtubule adsorbed onto the glass surface. The lifetime of bound Cy3-nucleotide in the absence or presence of the microtubule was 10 s or 0.08 s, respectively, showing that ATPase activity of the kinesin is strongly activated by microtubules. As the present system is equipped with a nanometer sensor, elemental steps of a single kinesin molecule can also be measured. By simultaneously measuring the individual ATP turnovers and elementary mechanical events of a single kinesin molecule, we will be able to obtain a clear answer to the fundamental problem of how the mechanical events are coupled to the ATPase reaction. (C) 1997 Elsevier Science B.V.