Cryopreserved fertilized mouse ova were used to generate transgenic mice via micromanipulation. Five-DNA constructions were injected into a total of 1,052 cryopreserved ova, of which 683 (65%) survived the injection and were transferred into recipients. Of 35 recipients, 66% became pregnant and littered a total of 88 pups. As controls, these DNA constructions were also injected into 1,123 fresh ova, of which 744 (66%) survived and were transferred. Of 42 recipients, 79% became pregnant and littered a total of 167 pups. That is, 22% of fresh ova that were transferred developed into live pups, whereas only 13% of cryopreserved ova did so. Of the pups born, 42 of the 167 (25%) produced from fresh ova were transgenic, and 28 of the 88 (32%) produced from cryopreserved ova were transgenic. In terms of the injected ova that had been transferred, 5.6% of the 744 fresh and 4.1% of the 683 frozen ova developed into transgenic mice. These data indicate that the efficiency of production of transgenic mice from cryopreserved ova is close to that from fresh ova. That observation and the fact that cryopreserved ova allow more efficient utilization of animals suggest that cryopreserved ova can be used instead of fresh ova to produce transgenic mice.