Constitutively migrating malignant rat liver epithelial cells obtained by Ha ras transformation exhibit a fibroblastoid phenotypein vitro. The cells deposit the anti-adhesive extracellular matrix (ECM) protein tenascin into their ECM migration tracks. The serum-free medium conditioned by these constitutively migrating cells contains an epithelial migration-stimulating activity (eMSA) that is neither cell-type-, nor species-specific. This eMSA fractionates in the range of 30 to 50kDa and binds to Mono-Q, Mono-S, and with low affinity to heparin—Sepharose. The conditioned medium also induces the expression of the serine proteinase inhibitor PAI-1. Both migration and expression of PAI-1 are inhibited by cyclic AMP, as previously shown for the migration of the non-transformed liver epithelial cells induced by several growth factors that act through tyrosine kinase receptors. These results suggest that the eMSA might act through signal transduction pathways similar to those of the growth factors previously studied. It is postulated that the eMSA, through both autocrine and paracrine mechanisms, is at least partially responsible for the malignant phenotype of the transformants.