The aim of this study was to examine the effects of transforming growth factor (TGF) β1 on the phenotype and the biological behavior of pancreatic cancer cell lines with and without mutations in the TGF-β signaling pathway and to elucidate whether the Ras signaling cascade participates in mediating these effects of TGF-β1. TGF-β-responsive (PANC-1, COLO-357, and IMIM-PC1) and nonresponsive (CAPAN1 and IMIM-PC2) pancreatic cancer cell lines with activating mutations of the Ki-Ras oncogene were treated with 10 ng/ml TGF-β1 over time. Phenotypic alterations were studied by electron and phase contrast microscopy and by immunohistochemistry and expression analyses of differentiation markers. The influence of TGF-β on tumor cell scattering, migration, and invasion was determined. The role of the Ras-mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) cascade in mediating TGF-β-induced morphological and functional effects were studied by pretreatment with the MEK1 inhibitor PD 98059 and by measuring ERK2 activation using immune complex kinase assays. TGF-β1 led to a reversible and time-dependent epithelial-mesenchymal transdifferentiation (EMT) in TGF-β-responsive pancreatic cancer cell lines, characterized by a fibroblastoid morphology and an up-regulation of mesenchymal markers and a down-regulation of epithelial markers. EMT was associated with an increase in tumor cell migration, invasion, and scattering. In the responsive cell lines, TGF-β1 induced a moderate but sustained activation of ERK2. EMT, the concomitant changes in gene expression, and the invasive and migratory potential were reduced or abolished by pretreatment with the selective MEK1 inhibitor. Thus, in TGF-β-responsive pancreatic cancer cells with activating Ki-Ras mutations, TGF-β1 treatment caused an EMT associated with a more invasive phenotype. Cross-talk with the Ras-MEK-ERK-signaling cascade appears to be essential for mediating these effects of TGF-β1.