Mucin O-glycosylation is initiated by a transfer of N-acetyl-D-galactosamine (GalNAc) to Ser and Thr residues in polypeptides with a family of UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferases (pp-GalNAc-Ts). In this paper, four human pp-GalNAc-Ts (pp-GalNAc-T1, T2, T3, and T4) were tested for their preferential orders of GalNAc incorporation into FITC-PTTTPITTTTK, a portion of the tandem repeat of human MUC2. The products were separated by reverse-phase HPLC and characterized by MALDI-TOF MS and peptide sequencing. pp-GalNAc-T1 showed preference for acceptor sites, but the order of the incorporation into these sites seemed to be random. In contrast, the GalNAc incorporation by pp-GalNAc-T2, T3, or T4 was not only site-specific but also according to the specific orders. Furthermore, pp-GalNAc-T2, T3, or T4 had distinct maximum numbers of GalNAc incorporations into this peptide.