Sporeamicin A is a 14-membered macrolide antibiotic produced by Saccharopolyspora sp. L53-18, which has strong antibacterial activity against Gram-positive bacteria. A new minor component designated sporeamicin B was discovered from the culture filtrates of the sporeamicin A-producing strain. Fermentation was carried out at 28°C for 161 hours in a 250-liter fermenter containing a medium consisting of glucose 3%, corn steep liquor 1%, dry yeast 0.6%, cobalt chloride 0.001% and FS-antifoam 028 0.04% (pH 7.0). The culture broth (200 liters) was filtered and the filtrate was extracted with ethyl acetate at pH 9.0. Isolation of sporeamicin B was accomplished using the general procedure for basic macrolide antibiotics and purified by precipitation, silica gel column chromatography and preparative reverse-phase HPLC. Sporeamicin B is basic, soluble in methanol, ethanol, acetone, ethyl acetate, benzene, chloroform and acidic water, but barely soluble or insoluble in n-hexane and water. It gave positive color reactions with potassium permanganate, iodine, Dragendorff and Molisch reagents, but negative with ninhydrin and Sakaguchi reagent. Its molecular formula was determined to be C36H61NO12 based on FAB-MS ((M+H)+, m/z 700) and elemental analysis. UV spectrum suggested an enone function (276 nm). IR spectrum showed enone (1620, 1690 cm⁻¹), ester carbonyl (1740 cm⁻¹) and hydroxyl (3450 cm⁻¹) functions. Structure determination via CI-MS and NMR data indicated that sporeamicin B has a structure where the neutral sugar cladinose of sporeamicin A is replaced by mycarose. Sporeamicin B exhibited antibacterial activity against Gram-positive bacteria, which was about two fold less than that of sporeamicin A.