The acceptor substrate specificities of ST6GalNAc I and II, which act on the synthesis of O-Linked oligosaccharides, were reexamined using ovine submaxillary mucin, [Ala-Thr(GalNAc)-Ala](n) polymer (n = 7-11). It has been suggested that only ST6GalNAc I can synthesize carbohydrate structures of siafyl-Tn-antigen; i.e., NeuAc alpha 2-6GalNAc-O-Thr/Ser [Kurosawa et al., J. Biol. Chem. 269, 19048-19053 (1994)] based on the result that ST6GalNAc I, not ST6GalNAc II, exhibited activity toward asialoagalacto-fetuin. In this study, me present evidence that both ST6GalNAc I and a exhibit activity toward asialo-OSM (ovine submaxillary mucin) and [Ala-Thr(GalNAc)-Ala](n) polymer (n = 7-11) which have only the GalNAc-O-Thr/Ser-structures. These results strongly indicate that not only ST6GAlNAc I but also LT are candidates for sialyl-Tn synthases.