A cytosolic fraction of human small intestine was prepared. It contained esterase activity toward N-alpha-acetyl-lysine-methyl ester and amidolytic activities toward substrates S-2238, S-2288 and S-2251. In addition there was present a plasminogen activator activity which could cleave plasminogen to produce plasmin and the plasmin hydrolysed the same chromogenic substrates. Plasmin generation was also followed by a time-dependent hydrolysis of 125-I labeled plasminogen or monitored by fibrin-agar plate. The plasminogen activator was related to urinary urokinase immunologically. Anti-urokinase IgG cross-reacted with cytosolic fraction in double immunodiffusion. When the cytosolic fraction was electrophoresed in discontinuous polyacrylamide gel, two regions of hydrolytic activity toward the urokinase-specific substrate S-2444 were found. The activity of one of these regions could be completely inhibited by anti-urokinase while the other was not. The plasminogen activator was partially purified by ammonium sulfate precipitation and Concanavalin A-bound Sepharose chromatography.