In vitro fertilized mouse eggs (C57BL/6N), followed by ultrarapid freezing were used for production of transgenic mice by microinjection of the chicken beta-actin promoter-driven the firefly luciferase cDNA (beta act-Luc). Following micromanipulation, the survival rates of the cryopreserved eggs and of the fresh in vitro fertilized eggs (control) were 70.8% (131/185) and 71.9% (159/221), respectively. After transferring them into oviducts of psudopregnant recipients on Day 1, 13.6% (17/125) of the cryopreserved eggs developed to live offspring and 14.1% (21/149) of fresh eggs did so. It was confirmed by Southern blotting analysis that each two transgenic mice were produced from the cryopreserved eggs (12%, 2/17) and the fresh eggs (10%, 2/21). All of transgenic mice produced from both eggs showed the expression of the luciferase gene. These results indicate that the in vitro fertilized eggs cryopreserved by ultrarapid freezing, can be, easily and conveniently, used for generation of transgenic mice.