We have studied the F-actin network in cycling suspension culture cells of carrot (Daucus carota L.) using rhodaminyl lysine phallotoxin (RLP). In addition to conventional fixation with formaldehyde, we have used two different nonfixation methods before adding RLP: extracting cells in a stabilizing buffer; inducing transient pores in the plasma membrane with pulses of direct current (electroporation). These alternative methods for introducing RLP revealed additional features of the actin network not seen in aldehyde-fixed cells. The three-dimensional organization of this network in nonflattened cells was demonstrated by projecting stereopairs derived from through-focal series of computer-enhanced images. F-actin is present in interphase cells in four interconnected configurations: a meshwork surrounding the nucleus; thick cables in transvacuolar strands and deep in the cytoplasm; a finer network of bundles within the cortical cytoplasm; even finer filaments that run in ordered transverse array around the cell periphery. The actin network is organized differently during division but it does not disappear as do the cortical microtubules. RLP stains a central filamentous cortical band as the chromatin begins to condense (preprophase); it stains the mitotic spindle (as recently shown by Seagull et al. [Seagull, R. W., M. Falconer, and C. A. Weerdenburg, 1987, J. Cell Biol., 104:995-1004] for aldehyde fixed suspension cells) and the cytokinetic apparatus (as shown by Clayton, L., and C. W. Lloyd, 1985, Exp. Cell Res., 156:231-238). However, it is now shown that an additional network of F-actin persists in the cytoplasm throughout division associating in turn with the preprophase band, the mitotic spindle, and the cytokinetic phragmoplast.