To identify and analyze acceptor sequences for O-glycosylation, we have developed an in vivo system expressing short peptides as glutathione S- transferase fusion proteins in the eukaryotic host Dictyostelium discoideum. Using this approach, we show that a short peptide motif (PTVTPT), present in the D. discoideum cell-surface glycoprotein PsA, is sufficient as a signal for O-glycosylation, even when fused to a heterologous protein. Monosaccharide analysis and solid-phase protein sequencing showed that the modification is a single N-acetylglucosamine attached to threonine residues. This was further confirmed by electrospray-mass spectrometry. The O-linked glycosylation of both this peptide and authentic PsA presents the modB- dependent carbohydrate-specific epitope identified by the monoclonal antibody MUD50. Substitution of threonine by serine residues in this peptide also yields a glycosylated fusion protein which is modified with single N- acetylglucosamine residues, but not all of the serines are glycosylated.