Expression vectors containing the pro-urokinase (pro-UK) cDNA (pSV2-proUK) and a dihydrofolate reductase cDNA (pSV2-dhfr or MMTV-dhfr) were cotransfected into CHO-dhfr- cells by the calcium phosphate precipitation technique. The dhfr+ transformants were selected by fibrinolytic agarose plate assay. Two colonies, named CLF-14 and CLF-8, exhibited significantly high expression levels of the biological activity of urokinase-type plasminogen activator (mu-Pa). They reached more than 24 IU/10(6) cells/48 h and 16 IU/10(6) cells/48 h, respectively. Examination of the cell supernatants for mu-Pa antigenicity using ELISA method also showed strong positive results, and the quantities of expression were about 0.14-0.22 micrograms/10(6) cells/48 h and 0.08-0.14 micrograms/10(6) cells/48 h, respectively. The mu-Pa secreted by stable transformed cells could be completely inhibited by UK anti-serum, but not by tissue-type plasminogen activator (t-PA) antiserum nor by normal rabbit serum.