Micro-plasminogen (mu Plg) gene amplified from human liver cells by reverse transcription PCR was inserted into expression vector pET-28a (pET-28a/mu Plg) and transformed into E. coli strain BL21(DE3). Recombinant human micro-plasminogen (rh-mu Plg) was over-expressed as inclusion bodies when induced with IPTG. After renaturation and purification, 16 mg rh-mu Plg/l was obtained with a homogeneity of 95% (w/w). Pro-urokinase (proUK)-induced rh-mu Plg activation was significantly faster than when Glu-plasminogen was the substrate. The catalytic efficiency of urokinase (UK) activation of rh-mu Plg was twice that of Glu-plasminogen. While recombinant human micro-plasmin (rh-mu Plm) and Lys-plasmin had a similar amidolytic activity against a small substrate, D-valyl-L-leucyllysine-p-nitroaniline dihydrochloride, Lys-plasmin activated proUK with a catalytic efficiency about fourfold greater than did rh-mu Plm. These results suggested that the kringle 1-5 domain of plasminogen and plasmin may modify both UK activation of plasminogen and plasmin activation of proUK, respectively.