DE-310 is composed of the topoisomerase-I inhibitor DX-8951 (exatecan) and carboxymethyldextran polyalcohol (CM-Dex-PA) carrier, which are covalently linked via peptidyl spacer (Gly-Gly-Phe-Gly). In this study, we investigated relationship between the cathepsin activity and the drug release of DE-310 by use of human liver origin cathepsin (B, L and H) and tumor cells (murine tumor cells (Meth A and M5076), and human tumor cells (HCT116, A549, PC-12, T98G, and HL-60)). Preliminary studies indicated that human liver cathepsin B produced Glycyl DX-8951 (G-DX-8951) from DE-310 more preferentially than DX-8951, whereas human liver cathepsin L produced DX-8951 preferentially. Release of drugs from DE-310 and cathepsin activities were measured in tumor cell types. The release of both DX-8951 and G-DX-8951 from DE-310 correlated well with cathepsin B activity of tumor cells. The release of DX-8951 was weakly, but not significantly, correlated with cathepsin L activity. In M5076 (high cathepsin activity) or Meth A (low cathepsin activity) xenograft models, the levels of DX-8951 and G-DX-8951 in M5076 were higher than in Meth A after single intravenous administration of DE-310. Our findings suggest that cathepsin B is primarily responsible for drug release from DE-310 in tumor.