Abstract The antitumor drug methanesulfonyl-m-anisidine, 4′-(9-acridinylamino) monohydrochloride (AMSA) (Cain9s acridine, NSC 141549), causes a limited, partially reversible decrease in size of the DNA of mouse L1210 leukemia cells as analyzed by centrifugation of cell lysates on alkaline sucrose gradients. Exposure of cells for 30 min to AMSA, 2.5 µg/ml, in tissue culture or to 150 µg/mouse in vivo results in a shift of long-term prelabeled DNA from >170S fractions to a broad band of about 30S. Higher doses or longer exposure times do not produce DNA much smaller than 30S. Labeled AMSA cosediments with heavy DNA in neutral sucrose gradients in which no degradation is observed, but AMSA is dissociated from both heavy and 30S DNA in alkaline sucrose gradients and appears only at the top of the gradient. On neutral gradients degradation to 30S DNA after AMSA exposure is detected only when cells are previously lysed in an alkaline medium but not when they are lysed in a neutral 70% formamide solution or when the neutral lysate is heated to 83° before centrifugation. Treatment of L1210 cells with other DNA intercalating agents (ethidium bromide, acridine orange, and phosphine orange) caused a similar degradation of the DNA on alkaline sucrose gradients. The results are interpreted to indicate that AMSA causes alkali-sensitive lesions of DNA at a limited number of sites; this effect correlates with the antitumor action of the drug and with other work showing long-term damage to chromosome structure.