A novel antitumor compound, N-beta-dimethylaminoethyl 9-carboxy-5-hydroxy-10-methoxybenzo[a]phenazine-6-carboxamide sodium salt (NC-190), has potent antitumor activity against in vivo and in vitro tumor models. In this study, we evaluated the cell-cycle effect of NC-190 on cultured HeLa S3 cells using DNA/bromodeoxyuridine(BrdU) bivariate flow-cytometric analysis. Continuous treatment with NC-190 for 72 h inhibited the growth of cultured HeLa S3 cells in a concentration-dependent manner. Its 50% growth-inhibitory concentration (IC50) was 0.039 micrograms/ml (0.085 microM). The cell-cycle effects of NC-190 were dependent on the drug concentration and the treatment period. Continuous treatment with a low concentration (0.1 micrograms/ml) of NC-190 inhibited cell-cycle progression from the G2 to the M phase, resulting in G2 accumulation. With increasing concentration, NC-190 delayed cell-cycle traverse in the S and G2 phases. At a higher (10 micrograms/ml) concentration of NC-190, cell-cycle traverse was prevented in the G1, S, and G2 phases. Under such conditions, NC-190 increased the numbers of S0-phase cells (the cells with DNA content corresponding to that of S-phase cells, but without BrdU incorporation). Treatment for 2 h with NC-190 at 10 micrograms/ml induced the accumulation of cells in the G2 phase, and cell debris was observed at 48 and 72 h after drug treatment. During this time, the proportion of cells in the S0 phase increased up to 19.2%. The colcemid-induced mitotic cell accumulation was delayed by NC-190 at a concentration of 0.1 micrograms/ml at 4 h after the addition of the drugs. The addition of higher concentrations (1 and 10 micrograms/ml) of NC-190 inhibited the increase in the mitotic fraction completely. These results indicate that the mechanism involved in the G2 arrest and the increment of S0-phase cells caused by NC-190 is associated with this compound-induced cell death.