The effects of aclacinomycin (ACM; NSC 208734) on cell viability, growth, and colony formation were investigated in suspension (Friend leukemia and L1210) and adherent (Chinese hamster ovary) cell systems. Cell cycle progression and the effect of the drug on various transition points in the cell cycle (i.e. G1 to S phase, through a window in early S phase and G2 phase to mitosis) were monitored by flow cytometry. Formation of Chinese hamster ovary cell colonies was inhibited by 50% following 24 hr of exposure to 0.05 micrograms ACM per ml whereas 1 hr of exposure to 1.0 micrograms ACM per ml reduced colony formation by only 30%. Stationary cultures required a drug concentration more than 5 times higher to reduce colony formation by an equivalent amount when present for 24 hr. Short-term (1-hr) exposure to drug concentrations up to 1.0 micrograms/ml had no effect on colony formation of stationary-phase Chinese hamster ovary cells. Cell growth was inhibited by 50% in suspension cultures of Friend leukemia and L1210 cells when exposed for 24 hr to 0.024 and 0.053 micrograms ACM per ml, respectively. Continuous drug exposure of Friend leukemia and L1210 cells to ACM concentrations of 0.05 to 0.1 micrograms/ml led to a slow down in cell progression manifested as an accumulation of cells in G2 + M phase by 24-hr and then in G1 phase by 48-hr culture. However, brief (1-hr) exposure of L1210 cells to 0.5 micrograms/ml resulted in an irreversible accumulation of cells in G2 + M phase. A more detailed examination of drug effects on the cell cycle determined that 0.1 micrograms ACM per ml resulted in a slow down in L1210 cells leaving G1 phase and entering mitosis and an accumulation of cells in G2 phase, although early S-phase cells appeared unaffected. At a 5 times higher drug concentration, exit of cells from G1 was almost completely halted, passage of cells through early S was slowed, and the entrance of cells into mitosis plateaued 3.5 hr after addition of the drug; G2-phase cells were only mildly affected. The RNA content of all cells examined was reduced by 35 to 50% depending upon dose and time of exposure. These findings are discussed in terms of the known biochemical effects of ACM on RNA and protein synthesis.