We reported previously that a monoclonal antibody, 6G9, raised against bovine submaxillary mucin (BSM) reacted with mucinous ovarian cancer and recognized tumor-associated sialylated carbohydrate antigens. To obtain structural information on the carbohydrate antigens recognized by 6G9, the reactivity of several mucins and carbohydrates with the antibody was determined by ELISA. Exoglycosidase digestion of BSM showed that 6G9 recognized Sia as a nonreducing monosaccharide but neither Gal nor GlcNAc. Reactivity of BSM with 6G9 decreased markedly on de-O-acetylation of BSM in mild alkali, and O-acetyl Sia obtained from BSM reacted with the antibody, indicating the presence of O-acetyl groups on Sia in the epitope. A sialyl-Tn structure located in the epitope was also demonstrated by the findings that de-O-acetylated BSM retained weak but significant reactivity with 6G9 and that ovine submaxillary mucin, major sugar chains of which are sialyl-Tn, reacted with 6G9 stronger than de-O-acetylated BSM. Furthermore, weak reactivity of NeuAcalpha2 --> 6GalNAc prepared from BSM demonstrated that 6G9 recognized the sialyl-Tn structure, but the modification of Sia with O-acetyl groups was essential for the recognition. The failure of 9-O-acetyl NeuAc, synthesized chemically, to react with the antibody implied that 6G9 recognized sialyl-Tn with O-acetylation on Sia distinct from C-9 O-acetylation.