For the purpose of reasonable evaluation of the cell kinetics of RNA synthesis inhibitors, the flow cytometric technique using acridine orange staining for simultaneous measurement of cellular DNA and RNA content was applied in order to analyze the effects of actinomycin D (ACT-D) on cultured cancer cells (PC1). ACT-D 0.05 microgram/ml, a dose that produced little inhibitory effect on the growth curve, led to G1 accumulation after the appearance of G2M, and a decrease in RNA content was concomitantly observed at 48-72h, although the RNA level had nearly recovered to normal at 120 h. On the other hand, ACT-D 0.1 microgram/ml, a dose that produced a significant inhibitory effect, resulted in the initiation of G1-phase accumulation, a maximal peak of G2M accumulation and a decrease in RNA content at 48 h, after which there was a tendency toward G1-phase accumulation and a marked decrease in RNA content which continued for 120 h. Such a marked decrease in RNA content coincided with high values of the G1a X G1b ratio in the G1 phase. These results suggest that ACT-D has an inhibitory action on the cell cycle between G1a and G1b, accompanying a marked decrease in RNA content.