In our screening for antitumor antibiotics inhibiting the growth of resistant P388 leukemia cells, a streptomyces strain MG883-12F2 was selected. This strain produced streptonigrin (2) and a new analog identified as 10'-O-demethylstreptonigrin (1), and was classified as Streptomyces afbits. Streptonigrin is known to have strong antitumor activity against various tumors but extremely high toxicity; studies on its structure-activity relationships and synthesis of analogs with high antitumor activity and low toxicity have been conducted. This paper reports the isolation and structure determination of demethylstreptonigrin (1) using NMR and physico-chemical properties. Demethylstreptonigrin (1) showed weak antibacterial activity. The 50% inhibition concentrations of streptonigrin (2) and demethylstreptonigrin (1) on P388 leukemia cells in vitro were 0.025 μg/ml and 0.58 μg/ml, respectively; on adriamycin-resistant P388 cells, they were 0.01 μg/ml and 0.41 μg/ml, respectively.