The most established methods for development of transgenic animals are the microinjection of DNA into the fertilized eggs, but it is still a procedure of certain complexity and high cost. Therefore, the idea of using sperm as a vehicle to carry exogenous DNA into eggs is very attractive, and there have been some successful reports. Though the methods are rather simple they sometimes have low reproducibility. To improve the technique we transinfected the spermatozoa in testicular duct, not in vitro, to produce mice which expressed human tissue plasminogen activator (tPA) in mammary gland. The results demonstrated that: (1) 5 transinfected mice mated 10 female mice in 10 days after operation, (2) 79 founders were developed and 42 survived, (3) using PCR to detect foreign DNA integrated into the genome of founders, 7 out of 42 founders were positive (16.67%), (4) The expression level of tPA was 48-80 ng/ml in the milk of 5 PCR positive founders and (5) the foreign DNA integrated into the genome was detected in 2 out of 4 1st offspring by PCR technique.