The actinomycins form a family of more than 45 related heteromeric peptide antibiotics containing the same phenoxazinone chromophore but differing in the amino acid composition of the two peptidolactone side chains. Binding to DNA and intercalation into the polynucleic acid double strand has been shown as the main activity of actinomycin D containing identical peptidolactone moieties, however, few information is available about DNA-binding activities of the other naturally occurring representatives of the actinomycin family. In the course of our screening for new microbial metabolites we disclosed Streptornyces sp. HKI-0155 as the producer of an orange pigment which displayed moderate antibacterial activity against Bacillus subtilis ATCC 6633. Due to m/z 1295.3 ([M+Na]+) in the ESI-MS spectrum and database searches suggesting the novelty of the metabolite we started an isolation program for this compound. The evaporated ethyl acetate extract of 10 liters of a 96 hours culture of Streptornyces sp. HKI-0155 was subjected to column chromatography on silica gel 60 (elution by n-hexane, CHCl3/MeOH 9 : 1 v/v). A yellowish fraction was further purified by preparative HPLC on silica gel RP18 using a gradient of water to 83% acetonitrile followed by an isocratic run, yielding 35mg of compound 1. UV and IR spectroscopic data suggested the similarity of the chromophore of 1 with that of the actinomycins. The chemical formula C61H84N12O18 was inferred by HRESI-MS (m/z 1295.5894 ([M+Na]+), calcd.: 1295.5924). FAB-MS and ESI-MSn experiments indicated the presence of N-methylvaline and sarcosine. Conclusive evidence for the structure of 1 was furnished by 1D and 2D NMR spectroscopy, which displayed 12 amide carbonyls and an additional signal at 178.16 ppm attributable to C-3 of the phenoxazinone chromophore.