In the course of our screening program for new antitumor antibiotics, we have previously reported on new antibiotics, duocarmycins A(1), Q (2) and C2 (3). Medium modification has produced two new antitumor antibiotics duocarmycins B₁ (4) and B₂ (5) in the fermentation broth of Streptomyces sp. DO-89. In this paper, the production, isolation, physico-chemical properties and biological properties of duocarmycins B₁ (4) and B₂ (5) are reported.