O-Glycosylation of three consecutive Thr residues in a fluorescein-conjugated peptide PTTTPLK - which mimics a portion of mucin 2 - by four isozymes of UDP-N-acetylgalactosaminyltransferases (pp-GalNAc-T1, T2, T3, or T4) was investigated. Partially glycosylated versions of this peptide, PT*TTPLK, PTTT*PLK, PT*TT*PLK, PTT*T*PLK, PT* degrees TTPLK, and PTTT* degrees PLK (*, N-acetylgalactosamine; degrees, galactose), were also tested. The products were separated by RP-HPLC and characterized by MALDI-TOF MS and peptide sequencing. The first and the third Thr residues act as the peptide's initial glycosylation sites for pp-GalNAc-T4, which were different from the sites for pp-GalNAc-T1 and T2 (the first Thr residue) or T3 (the third Thr residue) shown in our previous report. All pp-GalNAc-T isozymes tested exhibited distinct specificities toward glycopeptides. The most notable findings were: (a) prior incorporation of an N-acetylgalactosamine residue at the third Thr greatly enhanced N-acetylgalactosamine incorporation into the other Thr residues when pp-GalNAc-T2, T3, or T4 were used; (b) the enhancing effect of the N-acetylgalactosamine residue on the third Thr was completely abrogated by galactosylation of this N-acetylgalactosamine; (c) prior incorporation of an N-acetylgalactosamine at the first Thr did not have any enhancing effect; (d) pp-GalNAc-T2 was unique as it transferred N-acetylgalactosamine into the second Thr residue only when N-acetylgalactosamine was attached to the third one.