High concentrations of progesterone (PG) and mifepristone (MF) reinforce the effect of each other, reducing the endometrial cancer (Ishikawa) cell densities in vitro. Whether this effect is caused by cell cycle retardation, induction of apoptosis, or a combination of both was questioned. During a 5 days period in the absence of PG, the G(1)/G(0) fraction increased from 60% to 82% . After the first day of exposure with 32 and 95 muM PG the respective G(1)/G(0) fractions increased to 79% and 82% . A similar pattern was evident after exposure to MF. After the first day, MF caused a clear increase in the G(1)/G(0) fraction from 53% (control) to 72% (70 microM MF). In a third series of experiments, PG and MF were combined. After the first day, the G(1)/G(0) fractions were 50% in absence of active agents, 67% in presence of 32 microM PG, 66% in presence of 23 microM MF and 76% when PG (32 microM) and MF (23 microM) were combined. Both PG and MF induced apoptosis, which showed a time- and concentration-dependent pattern. In the control series the % apoptotic Ishikawa cells increased 2 to 3-fold during the experimental period and the effect of PG on apoptosis developed to a maximum after 4-5 days. On the fifth day, the trend was identical in three different assays commonly used to quantify different stages of apoptosis. The fractions of apoptotic cells, in the presence of 70 microM PG, increased from 2.5% to 28.2% (JC-1), from 1.4% to 13.2% (Annexin-FITC) and from 2.8% to 27.7% (DNA fragmentation assay), respectively. After five days, in a separate experiment, the fraction of apoptotic cells, as determined by JC-1 assay was 3.2% in the absence of active agents, 5.0% in the presence of 32 microM PG, 7.3% in the presence of 23 muM MF, and 12.4% in the presence of both PG (32 microM) and MF (23 microM). The present study shows that supraphysiological PG concentrations and high pharmacological concentrations of MF cause cell cycle retardation and induce apoptosis. In combination, PG and MF mutually reinforce these effects.