This study was conducted to evaluate the effect of supplementation of human or murine leukemia inhibitory factor (hLIF or mLIF) on in vitro maturation medium (TCM-199) containing FCS or BSA, and mLIF on semi-defined maturation medium containing BSA with or without hormones and in two different culture systems (single or group) on in vitro maturation, fertilization and development of bovine oocytes. Immature bovine oocytes were matured in vitro in TCM-199 supplemented with hLIF or mLIF (0 or 1,000 U/ml) containing FCS or BSA (Experiment 1), and mLIF as a supplement (0 or 1,000 U/ml) in TCM-199 containing BSA with or without hormones (Experiment 2) for 24 h. In Experiment 1, hLIF and mLIF added to a maturation medium have no positive effect on maturation, fertilization or development rates. In Experiment 2, the proportion of oocytes matured in TCM-199 with hormones showed significantly higher normal and total fertilization rates (P<0.01) and cleavage rates (P<0.05) than without hormones. The proportion of oocytes matured in a group showed significantly (P<0.01) lower normal and total fertilization rates, and higher polyspermy rates than those matured singly. There was no positive effect of mLIF added to the maturation medium on in vitro maturation, fertilization or development of bovine oocytes. These results indicate that there was no stimulating effect of hLIF or mLIF added to in vitro maturation medium on IVM, IVF or development of bovine oocytes.