Gelatin zymograms revealed in human plasma a constant 66 kDa proteolytically active polypeptide. In most plasma samples other major proteolytic activities were seen at Mr 92,000, 130,000 and 225,000. All four proteases were Ca2+-dependent metalloproteases and bound quantitatively to gelatin-Sepharose. Immunoblotting results indicated that the 66 kDa protease was the human fibroblast gelatinase/type IV procollagenase and that the other three proteases were macrophage/granulocyte-derived gelatinase components. The 66 kDa protease did not bind to conA- nor lentil lectin-Sepharose allowing its separation from the 92, 130 and 225 kDa proteases. During the isolation procedure the plasma gelatinase/type IV procollagenase tended to form a proteolytically active spontaneous disulfide-bonded dimer and a 62 kDa component that could also be obtained by digestion with trypsin. The same polypeptide changes occurred also in stored preparations of the corresponding protease isolated from fibroblast culture medium while the freshly purified protein contained only the 66 kDa proform.