During the course of screening for new antibiotics from Nocardia, Nocardia mediterranei var. kanglensis 1741-64 was found to produce a new ansamycin antibiotic kanglemycin A, which is active against Gram-positive bacteria. This paper reports the isolation and structure of kanglemycin A. The fermentation broth of the producing strain was cooled, filtered, and the filtrate was extracted with ethyl acetate at pH 2. The extract was concentrated, washed with phosphate buffer, evaporated to a syrup, and chromatographed on silica gel. Active fractions were processed to obtain kanglemycin A as orange rod crystals (1 g), mp 156°C (dec), [α]D +315.4° (c 0.2, MeOH). The molecular formula C50H63NO19 was deduced from elemental analysis (C 60.63, H 6.53, N 1.46), electron impact (EI) and field desorption (FD)-MS (m/z 981 (M+)) and 13C NMR data. IR spectrum (KBr) showed bands at 3450 cm⁻¹ (NH/OH), 1783 cm⁻¹ (ester CO), 1640 cm⁻¹ (NHCO), 1700 and 1618 cm⁻¹ (naphthoquinone CO). UV absorption maxima were similar to rifamycin S, indicating the same chromophore. The structure was determined by X-ray crystallographic analysis, revealing a skeleton consisting of a naphthalenoid nucleus with a 5-membered ring, a 17-membered ansa chain, monoethyl ester of 2,2-dimethyl succinic acid, and β-O-(2,6-dideoxy-3-O-methyl-D-ribo-hexopyranosyl) (digitoxose) on the ansa chain. Kanglemycin A is an acidic lipophilic compound, soluble in acetone, ethyl acetate, chloroform, slightly soluble in methanol and ethanol, insoluble in petroleum ether. It exhibits potent antimicrobial activity against Gram-positive bacteria, with MIC values of 0.1~0.2 μg/ml against Streptococcus sanguis No. 10, 0.003~0.006 μg/ml against Streptococcus pyogenes A12, and 0.05 μg/ml against Streptococcus pneumoniae.