VISUALIZATION of single actin filaments by fluorescence microscopy(1) led to the development of new in vitro assays for analysing actomyosin-based motility at the molecular level(2-5). The ability to manipulate actin filaments with a microneedle(6,7) or an optical trap(8) combined with position-sensitive detectors has enabled direct measurements of nanometre displacements and piconewton forces exerted by individual myosin molecules. To elucidate how myosin generates movement, it is necessary to understand how ATP hydrolysis is coupled to mechanical work at the level of the single molecule. But the most sensitive microscopic ATPase assay available still requires over 1,000 myosin(9). To enhance the sensitivity of such assays, we have refined epifluorescence and total internal reflection microscopies to visualize single fluorescent dye molecules. We report here that this approach can be used directly to image single fluorescently labelled myosin molecules and detect individual ATP turnover reactions. In contrast to pre,viously reported single fluorescent molecule imaging methods, which used specimens immobilized on an air-dried surface(10-12), method allows video-rate imaging of single molecules in aqueous solution, and hence can be applied to the study of man types of enzymes and biomolecules.