The human 66 000 mol. wt. plasminogen activator (HPA66; tissue‐type plasminogen activator) has been purified from melanoma cells by a one‐step affinity method with a monoclonal antibody. HPA66 purified in this way consists mainly of a one‐polypeptide chain form with small amounts (15%) of a form containing two polypeptide chains held together by one or more disulphide bridges. The one‐chain form was converted to the two‐chain form by catalytic amounts of plasmin. During the conversion, the enzyme activity of HPA66, as measured by an [125I]plasminogen conversion assay and with a chromogenic substrate, increased linearly with the percentage of the two‐chain form. A linear regression analysis showed that all enzyme activity could be accounted for by the two‐chain form, while the one‐chain form had no measurable enzyme activity (detection limit approximately 5% of the activity of the two‐chain form). Together with previous findings of inactive proenzymes to murine and human approximately 50 000 mol. wt. (urokinase‐type) plasminogen activators, these findings indicate that plasminogen activators are generally formed from inactive one‐chain proenzymes which are converted to active two‐chain enzymes by limited proteolysis, thus demonstrating a third step in a cascade reaction leading to extracellular proteolysis.