Fostriecin is a novel antitumor antibiotic. In vitro studies showed that fostriecin inhibits DNA topoisomerase II (Topo II) catalytic activity, protein phosphatases involved with cell-cycle control and histone phosphatases. The relative contribution of these mechanisms to the antitumor activity has not been elucidated, but Topo II inhibition seems to be the major mechanism of action at in vitro cytotoxic fostriecin levels. Tumor cell lines with decreased Topo II content showed similar or increased sensitivity to fostriecin, compared to the parent cell lines. The reduced-folate carrier is probably responsible for the cellular uptake of fostriecin. The possible clinical consequences of these in vitro observations are discussed.