It has been shown to date that a combined treatment with microtubule and microfilament inhibitors alters the cytoskeletal organization of keratin intermediate filaments in cultured HeLa, fetal mouse epidermal, and epithelial PtK2 cells, although neither of these inhibitors alone is able to do so. In the present study, we found that disruption of microfilaments with cytochalasin B induced a remarkable reorganization of keratin filaments in cultured human keratinocytes, while disruption of microtubules with colchicine did not affect keratin filaments. Keratin filament organization in the presence of cytochalasin B demonstrated a network of connecting star-like knots or foci. These foci coincided with actin aggregates that were formed by depolymerization of actin filaments as studied by double immunofluorescence using antiactin and antikeratin antibodies. Under these conditions, no change in microtubule arrangement was observed. Our observations suggest that the stability and architecture of keratin filament organization may be supported with the microfilament rather than the microtubule cytoskeleton in cultured human keratinocytes.