The effect of low levels of ATP on actin filament bundles in PtK2 cells was investigated by using 2-deoxyglucose, together with either sodium cyanide, sodium azide, or 2,4-dinitrophenol. Three actin filament systems were examined: stress fibers, cleavage rings, and dimethyl-sulfoxide (DMSO)-induced actin bundles in the nucleus. Of the three, only stress fibers disassembled when the ATP production was inhibited. The disassembly progressed slowly with the cells losing all stress fibers after about 90 min, but remaining in flat interconnected sheets. Mitotic cells that had progressed as far as metaphase when inhibitors were added, assembled cleavage rings. The process of cytokinesis took place in these cells but at a rate 5 to 10 times slower than normal, and disassembly of the cleavage ring was inhibited after the completion of cytokinesis. DMSO-induced nuclear actin bundles did not disassemble in cells depleted of ATP even when DMSO was eliminated from the medium. The peripheral aggregates of contractile proteins present in these cells became redistributed, however, and the cells flattened in the low ATP environment when DMSO was removed. Nuclear actin bundles did not form in DMSO-treated cells if the ATP inhibitors were present for as little as 5 min prior to DMSO exposure. Thus, the three types of actin filament bundles are affected in different ways by low intracellular levels of ATP. Stress fibers are most sensitive and cleavage rings, the least.