A culture of the human epidermoid carcinoma HEp 3 produces a plasminogen activator of Mr = 53,000 which we have purified to apparent homogeneity from serum-free conditioned medium by the combination of immunoaffinity chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The highly purified protein has the following properties: 1) It is indistinguishable from urinary urokinase in electrophoretic mobility, in immunodiffusion, and in autoradiographically visualized tryptic peptide maps obtained from the 125I-labeled proteins. 2) The HEp 3 protein differs from urinary urokinase in the following respects: (a) although the apparent molecular weights of the two are identical (Mr = 53,000), the urinary enzyme consists of two polypeptide chains, whereas the HEp 3 protein is a single chain form. (b) Urinary urokinase can be labeled easily by incubation with radioactive diisopropylfluorophosphate but the HEp 3 protein cannot. (c) When assayed by the hydrolysis of a synthetic chromogenic peptide substrate, the HEp 3 enzyme has less than 1% of the catalytic activity of urinary urokinase. 3) On controlled exposure to plasmin, the HEp 3 protein is converted to an active enzyme that is identical with urinary urokinase in molecular weight, polypeptide chain composition, diisopropylfluorophosphate labeling, and specific catalytic activity. We conclude that the HEp 3 protein is a proenzyme that can be converted to active two-chain urokinase by plasmin, probably by a single proteolytic nick in the polypeptide chain.