This study was conducted to establish the optimal temperature condition before oocyte activation in B6D2 F1 mouse. In experiment 1, two embryo culture media (CZB vs KSOM) were evaluated for the development of activated mouse oocytes. Parthenogenetic embryos cultured in KSOM showed better blastocyst development than ones cultured in CZB(56.2% vs 81.0%, p<0.01). Two-hour of pre-incubation before activation significantly reduced the number of hatched blastocysts in KSOM (22.0% versus 8.8%, p<0.05). In experiment 2, recovered oocytes were pre-incubated at different temperature conditions before activation. The experimental groups were divided by 5 as follows. Group A: pre-incubation for 120 min at 37℃, Group B: pre-incubation at 37℃ for 90 min then at 25℃ for 30 min, Group C: pre-incubation at 37℃ for 60 min then at 25℃ for 60 min, Group D: pre-incubation at 37℃ for 30 min then at 25℃ for 90 min, and Group E: pre-incubation at 25℃ for 120 min before activation. Group A (67.6%) and B (66.7%) showed better development to the blastocyst stage than other groups (Group C: 50.0%, Group D: 49.2%, Group E: 33.3%, p<0.05). The present study indicates that the temperature before activation affects the development of B6D2 F1 mouse parthenogenetic oocytes and exposure to room temperature should be limited to 30-min when the oocytes are left in HEPES-buffered medium for micromanipulation.