Fourteen congeners of podophyllotoxin were evaluated for their abilities to induce DNA breakage and inhibit growth of A549 human lung adenocarcinoma cells. Among the congeners studied were VP16-213, VM26, alpha-peltatin, beta-peltatin, and picropodophyllotoxin. Alkaline elution methods were used to assess DNA break frequencies following 1-h exposure to different concentrations of the congeners. DNA breakage was dependent upon drug concentration and was detectable when cells were exposed for 1 h to concentrations of VM26 as low as 0.05 microM. DNA breaks formed rapidly in cells after addition of drug but increased little after 30 min of continuous exposure. Repair of drug-induced DNA breaks was equally rapid with repair of 90% of the breaks occurring within 1 h following removal of the drug. Relationships between the structures of the congeners and the resulting DNA breakage activities were obtained, which correlated well with the cytotoxicity. The data suggest that a free hydroxyl group at the 4'-position is essential for DNA breakage activity, epimerization at the 4-position of the podophyllotoxin rings enhances activity, glucosylation of the hydroxyl group at the 4-position diminishes activity, aldehyde condensation with the glucose moiety greatly enhances activity, and the structure of the group associated with the resulting acetal linkage influences DNA breakage activity. These studies present quantitative data supporting and expanding upon the structure-activity relationship first proposed by Loike and Horwitz [Loike, J. D., & Horwitz, S. B. (1976) Biochemistry 15, 5443-5448].