To compare the site specificity of O-glycosylation in lower and higher eukaryotes, we expressed human granulocyte/macrophage colony-stimulating factor (hGM-CSF) in the yeast Saccharomyces cerevisiae and in COS-1 cells. Analyses of specific hGM-CSF mutants secreted by yeast led to the conclusion that efficient O-glycosylation in yeast requires residues S9 and T10. However, only S9 is used as an attachment point for an extended O-glycosyl chain in a 15.5-kDa hGM-CSF form. A 14.5-kDa hGM-CSF form, secreted by yeast, appears substituted by single mannosyl residues at both positions S9 and T10, indicating that O-glycosylation at T10 inhibits extension of the O-glycosyl chain attached to S9. As in yeast cells, the addition of O-glycosyl chains to hGM-CSF secreted by COS-1 cells requires the presence of S9 and T10 residues. These results demonstrate that, inspite of different biosynthetic routes, the selection of O-glycosylation sites is similar between lower and higher eukaryotes.