In the present study we have investigated the in vitro antitumor effects of three benzopyrans, 6-isobutyryl-5,7-dimethoxy-2,2-dimethylbenzopyran (1), 7-hydroxy-6-isobutyryl-5-methoxy-2,2-dimethylbenzopyran (2) and 5-hydroxy-6-isobutyryl-7-methoxy-2,2-dimethylbenzopyran (3) isolated from Hypericum polyanthemum. The three compounds tested demonstrated potent growth inhibitory activity at 40 microg/ml (<25% control growth) in the NCI-H460, HT-29 and U-373MG human cell lines. Determination of cell cycle distribution demonstrated that the antiproliferative effect of the three benzopyrans could be associated to alterations in the cell cycle phase distribution. Treatment with the IC50 of the three compounds induced an arrested in S phase. Only in cells treated with compound 3 did the percentage of sub-G1 population increase up to 9%, suggesting that this compound induced more cell death than the others. Consistent with sub-G1 analysis, appreciable oligonucleosomal DNA fragmentation was only observed in the NCI-H460 cell line treated with compound 3. From these results it can be suggested that despite no differences among the cytotoxicity of the three compounds, it was observed that the mechanism of their antiproliferative effects appears to be different.