Fostriecin is a new antitumor antibiotic which is being developed further as an anticancer agent based on its marked activity in murine leukemias. Its mechanism of action, however, has thus far remained unknown. The present study demonstrates that fostriecin inhibits the catalytic activity of partially purified type II topoisomerase from Ehrlich ascites carcinoma. Under the experimental conditions employed, fostriecin completely inhibited the enzyme at 100 microM. A general kinetic analysis showed that fostriecin inhibited topoisomerase in an uncompetitive manner with a Ki,app of 110 microM and produced kinetics that were distinctly different from those of VM-26 which exhibited noncompetitive inhibition. Fostriecin did not cause DNA strand breaks in L1210 cells, suggesting that it did not stabilize a cleavable complex as do other known inhibitors of this enzyme. Fostriecin, however, did partially inhibit DNA strand breaks produced by amsacrine. An analysis by flow cytometry showed that L1210 cells exposed to 5 microM fostriecin for 12 hr caused a block in the G2 phase of the cell cycle. These studies thus suggest that the mechanism by which fostriecin produces its antitumor effects may be through inhibition of topoisomerase II and that the type of inhibition is markedly different from existing antitumor agents which inhibit this enzyme.