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The plasminogen activator from a human melanoma cell line was purified with immunoadsorption as a major step. The cells were cultured in the presence of aprotinin in order to avoid proteolysis. A three-step purification involved adsorption on antibodies to porcine tissue plasminogen activator before chromatographies on arginine- Sepharose and Sephadex G-150. All solvents contained Tween-80 (0.01 %) and, except for the last step, aprotinin. The final product had a specific activity of about 220000 lu/mg measured against the WHO urokinasestandard.The activator obtained has an apparent Mt, of 72000 and consists of single-chain molecules. Evidence was obtained that four different types of activator variants occur. First and known previously, the one- chain form can be protcotytically cleaved into a two-chain form. Secondly, both the one-chain and two-chain molecules exhibit two forms with molecular weight differences of about 3000 (possibly due to carbohydrate differences). Thirdly, the once-chain preparations contain two variants, each constituting about 50 % of the material and differing in length by three N-terminal amino acids. Finally, a possible positional microheterogeneity was detected. Digestion with plasmin yields the two-chain form with disulfide-bonded polypeptide chains, `A' and `B' (from the N-terminal and C-terminal parts, respectively). At the same time, the variability of the original N terminus is removed. T he A chain keeps the two Mr variants (now about 40000 and 37000, respectively). The B chain (Mt about 33 000) contains the activcsite of the molecule, as demonstrated by labelling with [³H]diisopropyl phosphof1uoridate and is homologous to the enzymaticatly active chains of thrombin, plusmin and other serinc proteases. in contrast to these enzymes, the plasminogen activator is enzymatically active in the one-chain form. A speculative explanation for this activity may possibly be the presence of an c-amino group of a lysine residue at a position close to the bond cleaved in the two-chain form. [ABSTRACT FROM AUTHOR]