The purpose of this study was to evaluate the role of three glycosidases (α-mannosidase – α-MAN, β-N-acetyloglucosaminidase – NAGASE, and β-galactosidase – β-GAL) during in vitro maturation of bovine oocytes. COCs were aspirated from different size follicles (Small–SF: 2–5 mm, Large–LF: 6–8 mm, Controls–CF: 2–8 mm) from abattoir ovaries and were matured and fertilized in vitro either in groups (exp1 and 2) or individually (exp3). All three glycosidase activities were significantly higher in the maturation medium (MM) of oocytes collected from LF compared to those collected from SF (exp2 and 3), although in the case of α-MAN in exp3 the difference was not significant. In oocytes collected from SF, significantly lower α-MAN was observed in the MM of those that did not cleave compared to those that developed to >6 cells 2 days pi (exp3). A positive relationship was observed between the ratio of grade BC oocytes from LF and a-MAN (exp1), between a-MAN and cleavage rate (exp2, p = 0.095) and between α-MAN and the cell number of embryos (SF - exp3). A negative relationship was observed between NAGASE and cleavage rate (LF - exp2). Our results indicate that during maturation COCs release NAGASE and consume β-GAL and that there is a relationship between glycosidases activity in MM and oocyte quality. (This work was financed by GSRT and ERDF, Greece, and French Ministry of foreign affairs, France.)