Full-grown Xenopus oocytes are arrested at the prophase of the first meiotic division in a G(2)-like state. Progesterone triggers meiotic resumption also called the G(2)/M transition. This event is characterized by germinal vesicle breakdown ( GVBD) and by a burst in phosphorylation level that reflects activation of M-phase-promoting factor ( MPF) and MAPK pathways. Besides phosphorylation and ubiquitin pathways, increasing evidence has suggested that the cytosolic and nucleus-specific O-GlcNAc glycosylation also contributes to cell cycle regulation. To investigate the relationship between O-GlcNAc and cell cycle, Xenopus oocyte, in which most of the M-phase regulators have been discovered, was used. Alloxan, an O-GlcNAc transferase inhibitor, blocked G(2)/M transition in a concentration-dependent manner. Alloxan prevented GVBD and both MPF and MAPK activations, either triggered by progesterone or by egg cytoplasm injection. The addition of detoxifying enzymes ( SOD and catalase) did not rescue GVBD, indicating that the alloxan effect did not occur through reactive oxygen species production. These results were strengthened by the use of a benzoxazolinone derivative ( XI), a new O-GlcNAc transferase inhibitor. Conversely, injection of O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino-N-phenylcarbamate, an O-GlcNAcase inhibitor, accelerated the maturation process. Glutamine: fructose-6-phosphate amidotransferase inhibitors, azaserine and 6-diazo-5-ox-onorleucine, failed to prevent GVBD. Such a strategy appeared to be inefficient; indeed, UDP-GlcNAc assays in mature and immature oocytes revealed a constant pool of the nucleotide sugar. Finally, we observed that cyclin B2, the MPF regulatory subunit, was associated with an unknown O-GlcNAc partner. The present work underlines a crucial role for O-GlcNAc in G(2)/M transition and strongly suggests that its function is required for cell cycle regulation.