The human glycoprotein MUC1 mucin plays a critical role in cancer progression. Breast, ovarian, and colon cancer cells often display unique cell-surface antigens corresponding to aberrantly glycosylated forms of the MUC1 tandem repeat. In this report, (15)N- and (13)C-labeled forms of a recombinant MUC1 construct containing five tandem repeats were used as substrates to define the order and kinetics of addition of N-acetylgalactosamine (GalNAc) moieties by a recombinant active form of the human enzyme UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase I (ppGalNAc-T1; residues 40-559). Heteronuclear NMR experiments were performed to assign resonances associated with the two serines (Ser5 and Ser15) and three threonines (Thr6, Thr14, and Thr19) present in the 20-residue long MUC1 repeat. The kinetics and order of addition of GalNAc moieties (Tn antigen) on the MUC1 construct by human ppGalNAc-T1 were subsequently dissected by NMR spectroscopy. Threonine 14 was shown to be rapidly glycosylated by ppGalNAc-T1 with an initial rate of 25 microM/min, followed by Thr6 (8.6 microM/min). The enzyme also modified Ser5 at a slower rate (1.7 microM/min), an event that started only after the glycosylation of Thr14 and Thr6 side chains was mostly completed. Ser15 and Thr19 remained unglycosylated by ppGalNAc-T1. Corresponding O-glycosylation sites within all five tandem repeats were simultaneously modified by ppGalNAc-T1, suggesting that each repeat behaves as an independent substrate unit. This study demonstrated that the hydroxyl oxygens of Thr14 and to a lesser extent Thr 6 represent the two dominant substrates modified by ppGalNAc-T1 within the context of a complex MUC1 peptide substrate. More importantly, the availability of defined isotopically labelled MUC1 glycopeptide substrates and the relative simplicity of their NMR spectra will facilitate the analysis of other transferases within the O-glycosylation pathways and the rational design of tumor-associated MUC1 antigens.