Acemannan, a β-(1→4)-linked acetylated mannan (mannose content, 97%) isolated from the leaves of Aloe barbardensis Miller, was subjected to methylation analysis according to Arnarp et al. to determine the position of O-acetyl groups. The resultant partially methylated acemannan was converted to alditol acetates and analyzed by GLC-MS. Peak fragmentations identified 1,4,5,6-tetra-O-acetyl-2,3-di-O-methyl-D-(1-²H)mannitol (10.61 min) and 1,2,3,4,5-penta-O-acetyl-6-O-methyl-D-(1-²H)mannitol (10.42 min), indicating O-acetyl groups at C-2/C-3 and C-6. The result was verified by the method of de Belder and Norrman, where free hydroxyl groups were protected with methyl vinyl ether and O-acetyl groups were replaced with O-methyl groups, and similar GLC-MS results were obtained. From the data, it is concluded that the endogenous acetyl groups in acemannan are located at C-2/C-3 and C-6 in a ratio of approximately 50:50.