An improved technique for fluorescent labelling of heavy meromyosin has made it possible to detect on a light microscopic level the cellular sites of actin localization. Rabbit heavy meromyosin (HMM) was labelled with fluorescein isothiocyanate so that the actin binding site was protected during the reaction. The specificity of fluorescent HMM binding to cellular actin was tested by using glycerinated myofibrils. Staining was most intense in the I-Bands, and decreased at the edges of the A-Band, where actin filaments overlap with myosin. No staining occurred in the H-Zones or in the Z-Bands. The fluorescent HMM could be removed by washing with a relaxing solution. Similar sarcomeric patterns were obtained when embryonic chick skeletal and cardiac muscle cells were stained with fluorescent HMM. Localized fluorescent staining was also observed in smooth muscle fibers, axons and growth cones of nerves, acrosomal caps of sperm, cleavage furrows of dividing cells and pseudopods of various motile cells, all of which are known to contain actin. In sessile cells, the actin was found predominantly in fibrous bundles. This pattern of actin localization was altered when the cells underwent cleavage or became motile. The relationship between the intracellular distribution of actin and its function in the cell is discussed.