CI-937 and CI-942 belong to a new class of DNA complexes, the anthra[1,9- cd]pyrazol-6(2 H)-ones (anthrapyrazoles), and are being further developed as antitumor drugs based on their curative properties against murine solid tumour models. The biochemical effects of these agents were studied in L1210 leukemia in relation to other clinically used intercalators. After a 1-hr exposure, CI-937 and CI-942 reduced the cloning efficiency of L1210 cells by 50% at 3.0 × 10 −8 and 1.5 × 10 −7 respectively. Based on an ethidium displacement assay, these drugs bound strongly to DNA, reducing the fluorescence of an ethidium-DNA complex by 50% at concentrations of 23 and 33 nM for CI-937 and CI-942 respectively. This was comparable to mitoxantrone at 15 nM, but much more potent than Amsacrine which required over 1.3 μM. A distinct property of the anthrapyrazoles was a much more potent inhibitory effect on whole cell DNA synthesis than on RNA synthesis. After L1210 cells were exposed to drug for 2 hr the concentration needed to inhibit DNA synthesis by 50% was 0.33 and 0.57 μM for CI-937 and CI-942, respectively, whereas 2.0 and 11.3 μM were required to inhibit RNA synthesis by the same extent. This was in contrast to Adriamycin and mitoxantrone which inhibited both activities equally at similar concentrations. It was apparent that the inhibition of these processes was not due to substrate depletion since intracellular ribonucleoside and deoxyribonucleoside triphosphates either remained constant or were elevated after a 2-hr exposure to 1 or 10 μM drug. A similar discriminatory effect was observed on DNA and RNA polymerase in permeabilized cells, and the inhibition of nucleic acid synthesis in this system could be reversed by exogenously added DNA. Since the high incidence of cardiotoxicity associated with the administration of anthracyclines has been related to the formation of reactive oxygen species, the ability of the anthrapyrazoles to augment superoxide dismutase sensitive oxygen consumption was observed in a rat liver microsomal system. CI-937 and CI-942 induced 5- and 10-fold less oxygen consumption than Adriamycin, producing rates of 12.4, 24.2 and 138.9 nmoles/min/mg microsomal protein, respectively, at a drug concentration of 0.5 mM. The anthrapyrazoles were similar to other known intercalators in that they caused both single- and, to a lesser extent, double-strand DNA breaks that were tightly associated with protein in a concentration-dependent manner. The rates at which these lesions formed, however, were quite different between drugs. CI-937 at a concentration of 1 μM produced maximum strand breakage after 30 min, whereas CI-942 was much slower requiring at least 3 hr. If L1210 cells were treated with the anthrapyrazoles such that substantial strand breaks occurred and the drug removed, the breaks were repaired very slowly over the first 30 min but then additional lesions occurred thereafter for at least 2 hr. Additional DNA damage after drug was removed was also observed with Adriamycin and mitoxantrone but not Amsacrine with which 90% of the strand breaks disappeared within 30 min.