The site-specific glycan heterogeneity of human urinary erythropoietin was investigated by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Owing to the small amount of protein available, a strategy combining optimal sensitivity and specificity was used. Erythropoietin was reduced, S-alkylated and digested with endoproteinase Lys C. The peptides were separated by reversed-phase high-performance liquid chromatography and the molecular masses of the peptides determined by MALDI-MS. The peptides were identified by comparing the experimental masses with the masses predicted from the cDNA derived amino acid sequence. Glycopeptides were identified from the mass spectra based on the peak pattern caused by the glycan heterogeneity. They were further characterized after treatment with neuraminidase and endoproteases. All N-glycosylation sites exhibited fucose-containing complex-type glycans. The N-glycosylation sites at Asn38 and Asn83 are mainly occupied by tetraantennary glycans, whereas Asn24 is occupied by a mixture of bi-, tri- and tetraantennary glycans. A molecular mass glycoprofile for each glycosylation site was established based on the relative peak intensities observed in the MALDI mass spectra of the desialylated glycopeptides.