It has been reported that phosphatidylinositol 3-kinase (PI3K)-protein kinase B (PKB) pathway plays a crucial role in the meiotic resumption and progression to the metaphase II (MII) stage of oocytes. However, the role of this pathway in meiotic arrest at the MII stage (cytostatic activity) is not well understood. In this study, the effect of a PI3K inhibitor, LY294002, on the mitogen-activated protein kinase (MAPK) and p34^cdc2 kinase activities of matured porcine oocytes was examined. Immature oocytes were collected from ovaries and cultured in modified NCSU37 up to 48 hr. After culture, cumulus cells were removed and oocytes were cultured up to 24 h in medium supplemented with 25 or 50 μM LY294002. Groups of 10 or 20 oocytes were collected at each culture period for in vitro kinase assay of p34^cdc2 kinase and MAPK, respectively. Groups of 40 oocytes were also used for detection of PKB phosphorylation by Western blotting. After maturation culture, both the p34^cdc2 kinase and MAPK activities in the oocytes were gradually decreased in a time-dependent manner. Although 25 μM LY294002 did not affect either the p34^cdc2 kinase or MAPK activities, 50 μM LY294002 suppressed the PKB phosphorylation and slightly decreased MAPK activity, but not the p34^cdc2 kinase activity. Next, the effect of 10 μM Ca²⁺ ionophore which was reported as inducing a transient decrease of p34²⁺ kinase but not MAPK activities, was examined in LY294002-treated oocytes. Pronuclear formation of the oocytes was also evaluated by the aceto-orcein staining. By additional treatment with LY294002 after Ca²⁺ ionophore, both the MAPK and p34^cdc2 kinase activities were decreased in a time-dependent manner, concomitantly with improvement of pronuclear formation. Therefore, we concluded that PI3K is possibly involved in the maintenance of MAPK activity in matured porcine oocytes.