Dihydroxyanthraquinone, 1,4-dihydroxy-5,8-bis(( (2-[(2-hydroxyethyl)amino]ethyl)amino))-9,10-anthracenedione (NSC 279836), was observed to alter the cell cycle kinetics of a variety of mammalian cell lines as monitored by flow cytometry. Continuous exposure of Friend leukemia, L1210, and Chinese hamster cells to the drug in vitro at concentrations of 1.0 to 10 ng/ml resulted in the accumulation of cells in G2 by 24 hr in culture. When cells were exposed to dihydroxyanthraquinone for 30 min, washed free of drug, and cultured in fresh medium for 24 hr, a 10 times higher drug concentration was required to produce a G2 block identical to that observed during continuous exposure. Stimulation of human lymphocytes by phytohemagglutinin could be inhibited in a dose-dependent manner by brief pretreatment of cells with the drug. However, while previously stimulated but as yet noncycling lymphocytes were profoundly affected by much lower concentrations, proliferating lymphocytes were refractory to treatment with the drug up to a concentration of 1 microgram/ml. Exposure to the drug for 24 hr, at concentrations as low as 3.2 ng/ml, inhibited colony formation of exponentially growing Chinese hamster cells by 50%, whereas stationary culture required an 8-fold higher concentration to produce the same results. Drug concentrations in the range of 0.3 to 0.8 micrograms/ml over a period of 24 hr reduced the viability of Friend leukemia and L1210 cells by 50% as measured by trypan blue dye exclusion. In contrast, human lymphocyte viability was only mildly affected following 24 hr incubation with up to 5.0 micrograms dihydroxyanthraquinone per ml. There was a marked effect on cellular RNA content in two of the cell lines tested. Friend leukemia and L1210 cells blocked in G2 by the drug manifested a 140 and 70% increase in RNA content, respectively, when compared to control cells. In addition, though suboptimal concentrations of the drug resulted in a transient accumulation of cells in G2, optimal drug concentrations not only blocked cells in G2 but in the case of Friend leukemia and L1210 cells led to an increase in the proportion of cells with greater than 4C amounts of DNA. The results obtained with dihydroxyanthraquinone were compared to those obtained previously with a nonhydroxylated analog, anthracenedione (NCS 287513).