Camptothecins belong to a group of anticancer agents with a unique mechanism of action: poisoning of eukaryotic DNA topoisomerase I. Two camptothecin derivatives, topotecan (TPT) and CPT-11, are in clinical trials and their chemotherapeutic efficacy appears promising. Purpose : Our aim was to compare simultaneously the molecular and cellular pharmacology of the various camptothecin derivatives that are presently in clinical trials. Methods : Cytotoxicity of drugs toward human colon carcinoma HT-29 cells was determined by colony-forming assays. DNA single-strand breaks (SSB) were measured by alkaline elution. Drug potency to induce topoisomerase 1-mediated DNA cleavage and the sequence selectivity of the breaks were determined by sequencing gel autoradiography. Results : SN-38 and CPT were more cytotoxic than 9-AC and TPT, and CPT-11 was almost inactive toward HT-29 cells. IC 50 values were 8.8 nM for SN-38, 10 nM for CPT, 19 nM for 9-AC, 33 nM for TPT, and greater than 100 nM for CPT-11. In drug-induced DNA damage measured by alkaline elution drug concentrations producing 1000-rad-equivalents (C 1000 ), values were 0.037 μM for SN-38, 0.051 μM for CPT, 0.085 μM for 9-AC, 0.28 μM for TPT, and greater than 1 \xM for CPT-11. SN-38 remained the most potent compound in isolated nuclei, and CPT-11 was still inactive. The potency ranking was the same as in whole cells, and the C 1000 values were 0.0025 μM for SN-38, 0.012 μM for CPT, 0.021 μM for 9-AC, 0.44 μM for TPT, and greater than 0.1 μM for CPT-11. Potency difference between SN-38 and the other compounds was greater in isolated nuclei than in whole cells. Conclusions : Kinetics of the reversal of drug-induced SSB in isolated nuclei suggest that dissociation of SN-38 from cleavable complexes is much slower than that of CPT. Cleavage patterns of CPT and 9-AC were similar but differed from those of TPT and SN-38. Although in vitro analyses do not necessarily reflect chemotherapeutic efficacy, this study found that SN-38 is the most potent compound and that 9-AC and TPT are less active than CPT in this system. The effect of CPT-11 is minimal. Therefore, the clinical activity of CPT-11 may strongly depend on its hydrolysis to SN-38. Differences in DNA sequence selectivity and the stability of cleavable complexes induced by the drugs may also contribute to differences among CPT derivatives. [J NatI Cancer Inst 86: 836–842, 1994]