Two polyene macrolide, trichomycins A and B, were compared by physico-chemical and microbiological methods. The two antibiotics were found to have the same molecular formula, C58H84N2O18 (MW 1,096), by elemental analysis and FAB-MS. However, 1H and 13C NMR spectrometry studies indicated that the hydroxyl at C-5 of trichomycin A located on C-9 in trichomycin B. Trichomycin B possessed lower activities against fungi and yeasts than those of trichomycin A.