The potential of electrospray mass spectrometry (ESMS) for the sequencing of glycopeptides was evaluated using quadrupole time-of-flight (QTOF) technology in the MS/MS mode. The location of O-glycosylation sites was possible in the positive ion (+) mode by detection of prominent y- and b-fragment ions from the underivatized TAP25-2 [T1APPAHGVT9S10APDT14RPAPGS20T21APPA], an overlapping sequence of MUC1 tandem repeats which had been glycosylated in vitro by two GalNAc residues in the positions T9 and T21. The high mass resolution and accuracy of QTOF-(+)ESMS allowed reliable structural assignments. The reduced complexity of the fragment spectra and the higher signal-to-noise ratio render QTOF-(+)ESMS an alternative mass spectrometric approach to the identification of O-glycosylation sites when compared with sequencing by post-source decay matrix-assisted laser desorption/ionization MS. Diagnostic ions from the N-terminus in the b-series offered direct evidence, which was supported by indirect evidence from the C-terminus ions of the y-series. The higher glycosylated GalNAc2-substituted fragments were mainly observed as multiply ionized species.