In the course of our screening program for new antitumor antibiotics, we previously reported duocarmycins A, Q (2), and C2 (3). Medium modification led to the production of two new antitumor antibiotics, duocarmycins Bx (4) and B2 (5), in the fermentation broth of Streptomyces sp. DO-89. This paper reports their production, isolation, physico-chemical properties, and biological activities. Fermentation was conducted at 28°C for 100 hours in a medium containing maltose, dry yeast, Ebios, KBr, and other components. Isolation involved acidification, filtration, propanol extraction, and column chromatography on Diaion HP-20 and HP-20SS, yielding duocarmycin Bx (4) as a yellow powder and duocarmycin B2 (5) as an orange crystal. Their physico-chemical properties (melting point, specific rotation, elemental analysis, mass spectrometry, molecular formula, UV/13C NMR spectra, and solubility) are summarized. Both compounds have the molecular formula C26H26N3O8Br with a (M+H)+ ion at m/z 588. Antitumor activity against sarcoma 180 in mice (single intravenous dose) showed T/C ratios of 0.22 for duocarmycin Bx (4) at 0.5 mg/kg and 0.28 for duocarmycin B2 (5) at 0.25 mg/kg, comparable to mitomycin C (0.30 at 4 mg/kg). The LD50 values (intravenous, mice) were 0.37 mg/kg for Bx (4) and 0.28 mg/kg for B2 (5). Structural analysis revealed that duocarmycins Bx (4) and B2 (5) have a bromine atom substituting the chlorine atom in duocarmycins Q (2) and C2 (3), respectively. Treatment of duocarmycin A (1) with KBr-acetone produced duocarmycins Bx (4) and B2 (5) in a 1:4 ratio, suggesting duocarmycin A (1) is a precursor, and base treatment of duocarmycin B2 (5) regenerated duocarmycin A (1).