During the course of screening for new inducers of Friend mouse erythroleukemia cells, two active substances were isolated from the broth filtrate of Streptomyces diastochromogenes subsp. variabilicolor n. subsp. and identified as ansamycin antibiotics, named diastovaricins I and II. The producing organism was cultivated, and the antibiotics were isolated and purified via Diaion HP-20 column chromatography, EtOAc extraction, silica gel column chromatography, and Toyopearl HW40F column chromatography. Their physico-chemical properties (melting point, specific rotation, UV, IR, elemental analysis, FD-MS) were determined: diastovaricin I is red needles (mp 237-238°C, [α]21D +351° (c 0.1, MeOH), formula C39H45NO10, FD-MS m/z 688 (M+H)+); diastovaricin II is orange powder (mp 160-164°C, [α]21D +281° (c 0.1, MeOH), formula C44H52N2O12S, FD-MS m/z 833 (M+H)+). The structures were elucidated by 2-D COSY, C-H correlation spectral analysis, and LSPD experiments: diastovaricin I is 30-dechloro-30-hydroxynaphthomycin B, and diastovaricin II is its analog with the C-30 hydroxyl group replaced by an S-substituted N-acetylcysteine moiety. The effects on the induction of Friend cell differentiation were examined, showing that different concentrations affected cell number and the percentage of benzidinereactive cells. They exhibited no antimicrobial activity at 100 μg/ml and no toxicity in mice after intraperitoneal administration of 200 mg/kg.