The eukaryotic cell has evolved to compartmentalize its functions and transport various metabolites among cellular compartments. Therefore, in cell biology, the study of organization and structure/function relationships is of great importance. The cytoskeleton is composed of a series of filamentous structures, including intermediate filaments, actin filaments, and microtubules. Immunofluorescent staining has been most frequently used to study cytoskeletal components. However, it is also possible to fluorescently label isolated cytoskeletal proteins and either microinject them back into the cell or add them to fixed, permeabilized cells. Alternatively, it is possible to use the mushroom-derived fluorescinated toxins, phalloidin or phallacidin, to label F-actin of the cytoskeleton, as is described in this article. Phalloidin is available labeled with different fluorophores. The choice of the specific fluorophore should depend on whether phalloidin labeling for actin is part of a double-label experiment. In most cells, the abundance of actin filaments should provide a very strong signal. In double-label experiments, the fluorophore should be chosen to take this into account. In general, rhodamine labels are more resistant to photobleaching and can be subjected to the longer exposures required for finer structures.