The ability to measure properties of a single cross-bridge in working muscle is important because it avoids averaging the signal from a large number of molecules and because it probes cross-bridges in their native crowded environment. Because the concentration of myosin in muscle is large, observing the kinetics of a single myosin molecule requires that the signal be collected from small volumes. The introduction of small observational volumes defined by diffraction-limited laser beams and confocal detection has made it possible to limit the observational volume to a femtoliter (10(-15) liter). By restraining labeling to 1 fluorophore per 100 myosin molecules, we were able to follow the kinetics of approximately 400 cross-bridges. To reduce this number further, we used two-photon (2P) microscopy. The focal plane in which the laser power density was high enough to produce 2P absorption was thinner than in confocal microscopy. Using 2P microscopy, we were able to observe approximately 200 cross-bridges during contraction. The novel method of confocal total internal reflection (CTIR) provides a method to reduce the observational volume even further, to approximately 1 attoliter (10(-18) liter), and to measure fluorescence with a high signal-to-noise (S/N) ratio. In this method, the observational volume is made shallow by illuminating the sample with an evanescent field produced by total internal reflection (TIR) of the incident laser beam. To guarantee the small lateral dimensions of the observational volume, a confocal aperture is inserted in the conjugate-image plane of the objective. With a 3.5-mum confocal aperture, we achieved a volume of 1.5 attoliter. Association-dissociation of the myosin head was probed with rhodamine attached at cys707 of the heavy chain of myosin. Signal was contributed by one to five fluorescent myosin molecules. Fluorescence decayed in a series of discrete steps, corresponding to bleaching of individual molecules of rhodamine. The S/N ratio was sufficiently large to make statistically significant comparisons from rigor and contracting myofibrils.