Cytoskeletons of cultured cells prepared under mild conditions in the presence of "stabilization' buffer contain most of the fodrin present in the cells. The fodrin in these cytoskeletons was localized by immunofluorescence microscopy and found to be present in a cortical sheath of fine filaments. In general, the filamentous distribution showed no correspondence with actin bundles as revealed by double-label fluorescence microscopy. However, in cells with large and abundant stress fibers, some colocalization of fodrin with actin bundles was seen. Treatment of cells with either cytochalasin A or D caused disorganization of the actin bundles whereas fodrin still showed a filamentous distribution in cytoskeletons of the cytochalasin-treated cells. Implications of these results for the organization of the fodrin-containing sheath of cultured cells is discussed.