he effects of brief actinomycin D treatment (0.1 microgram/ml, 0.5 h) on inhibition of cell growth and colony formation were studied in synchronized HeLa cells. Cells in late S and G2 phases were found to be maximally sensitive to inhibition of cell growth and colony formation after short exposure to actinomycin D. Cells in G1 and early S phases were less responsive to brief actinomycin D treatment, although there was a slowdown of cell growth between 24 and 48 h after removal of actinomycin D, recovery of cell growth was observed late (> 48 h) after drug removal. Cells in mitosis were maximally resistant to brief actinomycin D treatment, and continued to grow as did the control cells without drug. The effects of actinomycin D on inhibition of cell growth and colony formation were abolished by novobiocin but not by aphidicolin present during a brief actinomycin D treatment of cells at various cell cycle stages. Our results suggest that the effect of actinomycin D is cell cycle phase-dependent and may be involved in the action of topoisomerase II. Furthermore, actinomycin D at a low dose (0.1 microgram/ml, 0.5 h) induced a slight G1 block while a brief exposure to high dose actinomycin D (1.0 microgram/ml, 0.5 h) caused a slowdown in the rate of cell progression through S and G2/M phases. Similar S and G2/M phase block was seen in cells that had been briefly treated with actinomycin D (0.1 microgram/ml; 0.5 h) during late S and G2 phases.