The O-linked glycosylation of the main acidic high-molecular-weight glycoprotein from ascites fluid from patients with ovarian cancer were analyzed. The O-linked oligosaccharides were shown to consist of mainly highly sialylated core 1 and 2 structures with a smaller amount of sulfated core 2 structures. These structures were shown to be able to be further extended into small keratan sulfate (KS)-type oligosaccharides with up to four N-acetyllactosamine units. Proteomic studies of the acidic fraction of ascites fluid from patients with ovarian cancer showed that this fraction was enriched in proteoglycans. Among them, lumican, agrin, versican and dystroglycans were potential candidates, with threonine- and serine-rich domains that could carry a significant amount of O-linked glycosylation, including also the O-linked KS. Glycomic analysis using liquid chromatography (LC)-tandem mass spectrometry (MS/MS) also showed that the disialic acid NeuAc-NeuAc- was frequently found as the terminating structure on the O-linked core 1 and 2 oligosaccharides from one ascites sample. Also, a small amount of the epidermal growth factor (EGF)-associated O-linked fucose structure Gal-GlcNAc-Fucitol was detected with and without sialic acid in the LC-MS/MS analysis. Candidate proteins containing O-linked fucose were suggested to be proteoglycan-type molecules containing the O-linked fucose EGF consensus domain.