Urinary metabolites of DX-8951 ((1S,9S)-1-amino-9-ethyl-5-fluoro- 1,2,3,9,12,15-hexahydro-9-hydroxy-4-methyl-10H,13H- benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione, CAS 171335-80-1, exatecan) in rats and humans were identified. Rats were dosed with the drug, and two major metabolites (UM-1 and UM-2) in the urine were isolated and purified by using ion-exchange column and HPLC. From NMR and mass spectra, they are suggested to be 4-hydroxymethyl metabolite (UM-1) and 3-hydroxy metabolite (UM-2) of the drug. Their chemical structures were confirmed by comparing their NMR spectra with those of chemically synthesized metabolites. Two major metabolites were found in human urine obtained in phase I trial. They were also confirmed to be UM-1 and UM-2 by LC/MS/MS by comparing their mass fragment patterns with those of synthetic metabolites.