Studies have established that cryoprecipitates of the plasma of tumor patients contain a biological activity enhancing morphological cell transformation (transformation-enhancing factor; TEF) in cultures of chicken embryo fibroblasts infected with temperature-sensitive mutants of Rous sarcoma virus. We report here that similar TEF activity is effected by defined fragments of human plasma fibronectin obtained by limited digestion with major humoral or tissue proteinases. TEF activity was obtained from plasminolytic fragments of fibronectin and from cathepsin G-treated fibronectin. No activity was recorded from intact dimeric fibronectin or its reduced and alkylated subunits, from fibrinogen or its plasminolytic fragments, or from plasmin (EC 3.4.21.7) or cathepsin G (EC 3.4.21.20) treated or untreated with proteinase inhibitors. All of the TEF activity of the proteolytic fragments of fibronectin was located on the gelatin-binding peptides. The minimum effective doses in the TEF assay were 750 ng/ml of plasmin-treated fibronectin, 100 ng/ml of gelatin-binding plasminolytic fibronectin (enriched in Mr 180,000--190,000 polypeptides), and 100 ng/ml of gelatin-binding fragments of cathepsin G-treated fibronectin (enriched in a Mr 30,000 fragment). TEF activity of proteinase-treated fibronectin was inhibited by gelatin and by intact dimeric fibronectin. The potent TEF activity of proteolytic fragments of fibronectin raises the possibility that they may have a role in malignant transformation.