Marine ascidians belonging to the family Didemnidae have proven a remarkable source of chemically diverse natural products with potent biological properties. As part of our continuing investigation of marine invertebrates from the Fiji Islands, we collected the thin encrusting orange ascidian Polysyncraton lithostrotum from Namenalala Island. A methanolic extract of the organism showed induction of SOS response in an agar based biochemical induction assay (BIA) and a similar cytotoxicity profile to calicheamicin in a 26 cell line human tumor panel, which were suggestive of a DNA cleavage agent. Bioautography directed fractionation of the extract using the BIA assay gave a new enediyne antitumor antibiotic namenamicin (1) in 10⁻⁴% yield (1 mg from 1 kg of frozen tissue). Namenamicin contains the same "enediyne warhead" as the calicheamicins; however, the attached carbohydrate moiety differs in replacement of the N-O sugar linkage between the A and B sugars with a C-O, an S-methyl substituent at the 4 position of the A sugar, and the absence of a benzoate ring appended to the B sugar. Namenamicin exhibited potent in vitro cytotoxicity with a mean IC50 of 3.5 ng/mL and in vivo antitumor activity in a P388 leukemia model in mice (ILS 40% @ 3 µg/kg). It also showed potent antimicrobial activity. DNA cleavage experiments indicated that namenamicin cleaved DNA with a slightly different recognition pattern than calicheamicin γ1 I. A molecular formula of C43H62N2O14S5 was suggested by a prominent ion in the electrospray mass spectrum at m/z 991, corresponding to (MH)+. Detailed analysis of 1H and 13C NMR spectra, a suite of 2D correlation experiments, and comparison with the calicheamicins and esperamicins readily allowed complete assignment of the aglycone. The sequence specific DNA interactions of namenamicin were mapped on a 142 base pair pBR322 restriction fragment and compared to calicheamicin γ1 I, revealing fewer high specificity cleavage sites and a slightly altered recognition pattern likely due to structural differences in the carbohydrate moiety.