Actin in cultured fibroblasts is organized into a complex set of fibers. Patterns of organization visualized with antibody to actin are similar but not identical to those visualized with fluorescein isothiocyanate-phalloidin (Fl-phalloidin), a chemical that binds to F-actin polymer with a dissociation constant of 2.7 X 10(-7) M [Wulf, E., Deboben, A., Bautz, F. A., Faulstich, H. & Wieland, T. (1979) Proc. Natl. Acad. Sci. USA 76, 4498-4502]. Fl-phalloidin reveals that transformed cells have fewer, finer, and shorter F-actin-containing structures than do normal cells. Two-color fluorescence microscopy of single cells reveals that F-actin staining by Fl-phalloidin picks out the cytoskeletal cables more sharply than does antibody to actin, due to a reduced intracellular background fluorescence. This improved resolution permits sorting of cellular Fl-phalloidin patterns into four classes ranging in organization from 90% of the cytoplasm occupied by large cables to the absence of detectable cables. Reproducible differences in pattern distributions between normal and transformed cell lines have been quantitated. Fl-phalloidin together with rhodamine-based indirect antibody to simian virus 40 tumor antigen reveals a direct relationship between the degree of pattern change and simian virus 40 nuclear antigen expression in intermediate transformed 3T3 cell lines [Risser, R. & Pollack, R. (1974) Virology 59, 477-489].