We observed the localization of the contractile proteins myosin, filamentous actin, alpha-actinin, tropomyosin, and vinculin in surface-activated, spreading human platelets using a single fluorescence staining procedure and conventional fluorescence microscopy. Myosin was distributed in a speckled pattern that extended radially from the granulomere. F-actin demonstrated cable-networks. Tropomyosin and alpha-actinin occurred in a punctuate distribution, and vinculin was localized at adhesion sites. Although myosin, F-actin, alpha-actinin, tropomyosin, and vinculin were not studied in resting platelets, our data support the idea that these contractile proteins are reorganized and reassembled in activated platelets during platelet function.