O-glycosylation of mucin is initiated by the attachment of N-acetyl-D-galactosamine (GalNAc) to serine or threonine residues in mucin core polypeptides by UDPGalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts). It is not well understood how GalNAc attachment is regulated by multiple ppGalNAc-Ts in each cell. In the present study, the expression levels of murine ppGalNAc-Ts (mGalNAc-Ts), T1, T2, T3, T4, T6, and T7 were compared between mouse colon carcinoma colon 38 cells and variant SL4 cells, selected for their metastatic potentials, by using the competitive RT-PCR method. The expression levels of mGalNAc-T1, T2, and T7 were slightly higher in the SL4 cells than in the colon 38 cells, whereas the expression level of mGalNAc-T3 in the SL4 cells was 1.5% of that in the colon 38 cells. Products of enzymatic incorporations of GalNAc residues into FITCPTTTPITTTTK peptide by the use of microsome fractions of these cells as the enzyme source were separated and characterized for the number of attached GalNAc residues and their positions. The maximum number of attached GalNAc residues was 6 and 4 when the microsome fractions of the colon 38 cells and SL4 cells were used, respectively. When the microsome fractions of the colon 38 cells were treated with a polyclonal antibody raised against mGalNAc-T3, the maximum number of incorporated GalNAc residues was 4. These results strongly suggest that mGalNAc-T3 in colon 38 cells is involved in additional transfer of GalNAc residues to this peptide.