Rifamycins and streptovaricins have potent antibacterial activities and contain an aliphatic ansa chain and naphthalene system as constituents. Several natural rifamycins have been found as the metabolites of Amycolatopsis mediterranei, and rifamycin W was isolated from a mutant strain of A. mediterranei as a biosynthetic intermediate. In the course of a chemical screening program, A. mediterranei var. kanglensis 1747-64 was found to produce a novel antibiotic, 31-homorifamycin W (1), belonging to the rifamycin family. This communication describes the fermentation, isolation, and structural elucidation of 1. Strain 1747-64 was cultured in an 80-liter jar fermentor with a production medium (glucose 5%, yeast meal 1.2%, peanut meal 0.5%, peptone 0.5%, soybean oil 3%, CaCO₃ 0.1%, pH 6.5) at 28°C for 86 hours, agitated at 250 rpm and aerated at 10 liters per minute. The broth filtrate was adjusted to pH 3 with 6N HCl, extracted with ethyl acetate, and the organic layer was concentrated. The residue was purified via silica gel column chromatography (chloroform-methanol, 5:1), Sephadex LH-20 column chromatography (methanol), and preparative TLC (chloroform-methanol-acetic acid, 100:10:1), yielding 30 mg of brown powder of 1. Physicochemical properties showed 1 is soluble in methanol, acetone, and ethyl acetate, slightly soluble in chloroform, and insoluble in water and hexane. Structural elucidation using HRFAB-MS gave a molecular formula of C₃₆H₄₇NO₁₁ ([M+H]⁺: calcd 670.3227, found 670.3257). UV spectroscopy indicated a chromophore similar to rifamycin W. IR spectroscopy identified amide carbonyl (1690 cm⁻¹), quinone carbonyl (1625 cm⁻¹, intramolecular hydrogen bond), and quinone chromophore (1495 cm⁻¹) groups. NMR spectroscopy (¹H, ¹³C, 2D COSY) confirmed the structure, with ¹H NMR spectra resembling other rifamycins and ¹H COSY establishing carbon connectivities including 34-CH₃-26-CH-25-CH-, 33-CH₃-24-CH-23-CH-, 32-CH₃-22-CH-, 31a-CH₃-31-CH₂-, and segments of the ansa chain. These data confirm 31-homorifamycin W as a new rifamycin derivative.