Dihydrof olate reductase (DHFR) deficient Chinese hamster ovary (CHO) cells were transf ected with expression plasmid constructed from human tissue-type plasminogen activator (t-PA) and DHFR gene. The transf ected cells were selected with a medium containing methotrexate (MTX, 0-5NM), followed by isolation of several DHFR+ clones. It was definitely obserbed that the expression levels of t-PA were dose dependently elevated on the MTX resistance and the gene copy number. This recombinant t-PA had a similar specific activity and the same N-terminal amino acid sequence to human melanoma cell derived t-PA, and also showed similar characteristics in a neutralization test with a human t-PA, SDS-PAGE, PAS staining, zymography and human plasma clot lysis time assay. The MTX-selected high expression clone could keep constant production of t-PA without MTX for at shortest 5 months. These results strongly suggestted that CHO cells-DHFR gene amplification system was useful for producing a large amount of human glycoprotein with a complicated structure such as t-PA.