As reported in a previous paper, we isolated two 1,4-diaminocyclitol-containing aminoglycoside antibiotics, istamycin A and B from culture filtrates of Streptomyces tenjimariensis nov. sp. as the major components. Istamycin A and B exhibited strong inhibition against Gram-positive and Gram-negative bacteria except pseudomonas. In this paper, we report the production of 2"-N-formimidoylistamycin A and B by Streptomyces tenjimariensis SS-939. The culture filtrate was passed through a column of Amberlite CG-50 (Type I, 70% Na+, 0.5 ml), eluted with 0.25 M H2SO4, concentrated, and analyzed by high performance liquid chromatography on a Waters' 206A Compact System with a Radial Pak C18 column (0.5×10 cm, 10 μm) and a Waters' Data Module 730 (mobile phase: 5% aqueous methanol containing 0.3 M Na2SO4 and 0.005 M PIC B7, flow rate: 1 ml/minute, detection: fluorescence with excitation at 340 nm and emission at 425 nm using a reagent of 0.05 M borax-0.006 M o-phthalaldehyde in 0.5% aqueous 2-mercaptoethanol, injection volume: 5~30 μl). Two major peaks at 8.71 and 10.50 minutes of retention time were observed instead of those of istamycin A (6.51 minutes) and B (7.75 minutes), which coincided with synthetic 2"-N-formimidoylistamycin A and B. This was confirmed by isolation of enough 2"-N-formimidoylistamycin A and B for chemical identification. Streptomyces tenjimariensis SS-939 was cultured at 27℃ for 24 hours on a rotatory shaker (180 rpm) in a 500-ml baffled Erlenmeyer flask containing 110 ml of a seed medium (2.0% starch, 0.2% glucose, 2.0% soybean meal, 0.2% sodium palmitate, 0.3% NaCl, 0.1% MgSO4·7H2O and 0.1% K2HPO4). The seed culture (1.5 ml) was inoculated to 110 ml of a production medium (6.0% corn meal, 2.0% wheat germ, 0.6% CaCO3 and 0.05% MgSO4·7H2O, pH 7.0) and cultured for 7 days at 27℃ on a rotatory shaker (180 rpm). The cultured broth (20 liters) from 182 flasks showed a bioactivity of 113 μg/ml against Bacillus subtilis PCI 219 using pure istamycin B (1,000 μg/mg) as the assay standard. After successive filtrations at pH 2.5 and 7.0, the antibiotics in the filtrate (17 liters) were adsorbed on a column of Amberlite IRC-50 (70% Na+, 15 liters) and eluted with 1 M HCl. Sodium p-toluenesulfonate (469 g) was added to the active eluate (2.9 liters, 437 μg/ml) as the organic counter ion, and the antibiotics in the solution (pH 5.0) were adsorbed on a column of Dialon HP-20 (1.8 liters). After washing with 10^-5 M HCl (3 liters), the column was eluted successively with 10^-5 M HCl in 5%, 7.5%, 10%, 12.5% and 15% methanol. Fractions (Nos. 2201~2550) containing 2"-N-formimidoylistamycin A were combined and concentrated to 1.0 liter (632 μg/ml), and fractions (Nos. 2551~3000) containing 2"-N-formimidoylistamycin B were concentrated to 1.1 liters (284 μg/ml). Purification of 2"-N-formimidoylistamycin A or B was achieved by Amberlite CG-50 (Na+) column chromatography eluted with 0.25 M H2SO4 followed by charcoal column chromatography eluted with 0.005 M H2SO4. The eluate was passed through a column of Amberlite IRA-400 (SO4^2-) and lyophilized to give pure 2"-N-formimidoylistamycin A (486 mg) or B (422 mg) as the disulfate. 2"-N-Formimidoylistamycin A disulfate trihydrate is a colorless hygroscopic powder, gradually decomposed at over 210℃, [α]19D +77° (c 1, water), bioactivity: 660 μg/mg of istamycin B. Anal. Calcd. for C18H36N6O5·2H2SO4·3H2O: C 32.42, H 6.95, N 12.60, S 9.62. Found: C 32.34, H 6.52, N 11.82, S 9.69. 2"-N-Formimidoylistamycin B disulfate tetrahydrate is a colorless hygroscopic powder, gradually decomposed at over 215℃, [α]19D +82° (c 1, water), bioactivity: 730 μg/mg of istamycin B. Anal. Calcd. for C18H36N6O6·2H2SO4·4H2O: C 31.58, H 7.07, N 12.27, S 9.37. Found: C 31.40, H 6.27, N 11.58, S 9.86. Minimum inhibitory concentrations of 2"-N-formimidoylistamycin A and B on a Mueller-Hinton medium are shown in Table 1. Intravenous injection of 200 mg (base)/kg of 2"-N-formimidoylistamycin A or B disulfate to mice caused no deaths. Spectral data and synthesis of these antibiotics will be reported in detail elsewhere.