In our preceding paper, we described the isolation and structure elucidation of antibiotic A83094A (16-deethylindanomycin) from the biomass of Streptomyces setonii. The culture filtrate from a fermentation of this same organism contains two broad-spectrum antibiotics which were isolated and determined to be 5-hydroxymethylblasticidin S (A83094B) and blasticidin S (A83094C). The flow diagram for their isolation as a complex is presented in Fig. 2, with antibiotic levels at each step determined by disc plate assay vs. Salmonella gallinarum and HPLC. Separation of the complex into individual antibiotics was accomplished using semi-preparative HPLC. 5-Hydroxymethylblasticidin S (A83094B) is a water soluble compound: MP>225°C (char); UV λmax (H2O, pH 7) 272nm. Its molecular weight of 452 (C18H28N8O6) was determined by fast atom bombardment (FAB)-MS data displaying a M+H+ at m/z 453 (a 30 dalton increase compared to blasticidin S) and a monosodiated ion at m/z 475. The 1H NMR of A83094B (500 MHz, DMSO-d6) compared to authentic blasticidin S indicates structural similarity, with differences including loss of 5-H in A83094B and addition of a hydroxymethylene at δH 4.21; a nuclear Overhauser effect (NOE) is observed between the new methylene and 6-H. The structure of A83094B is similar to Sch 36605.