In this study, the two forms of affinity-purified transformation-associated proteins (TAPs) (68 and 64 kD) were shown to have different substrate preferences. For the 68-kD TAP, the order of substrate preference was collagen types I, III, and V; fibronectin; gelatin; and collagen IV. For the 64-kD TAP, the order of substrate preference was collagen I, III, and V and gelatin. The 64-kD TAP did not cleave collagen IV and fibronectin. We also found a 71-kD metalloproteinase in the concentrated purified TAPs that reacted only weakly with a TAP monoclonal antibody and showed this substrate preference: collagen I, III, and V; gelatin; and collagen IV. Whether this 71-kD TAP is a new member of the rat metalloproteinase family will be investigated.