Short-term stimulation with insulinotropic factors may induce morphologic and functional changes in primary ductal cell cultures as a potential source of stem cells. We sought to assess the capacity of hepatocyte growth factor (HGF) to induce expression and maturation of proteins--PDX-1 and GLUT-2--and the subsequent beta-cell secretory profiles. HGF, which is involved in pancreatic development, may induce islet beta-cell neogenesis. Primary ductal cell monolayers were cultured in Click's + FBS 10% at 37 degrees C until tissue confluence. The medium was enriched with HGF (10 ng/mL for different periods); controls were treated for similar times with normal culture medium. At the end of the study, three-dimensional islet-like cell aggregates were observed in both conditions. In all conditions immunostaining studies showed positivity for the major endocrine-phenotype cell markers: insulin, PDX-1, glucokinase, and GLUT-2. Furthermore, treatment with HGF for short periods induced the expression of a functionally active, phosphorylated isoform of PDX-1. Finally, we observed that under basal conditions the cells initially and progressively released proinsulin throughout 5 days in all settings. Thereafter proinsulin was gradually replaced by insulin in the culture medium, reflecting a maturation progress. This pattern of insulin maturation and release was more evident when the cells were continuously stimulated with HGF for 12 days. The employed stimuli seemed to differentiate the original ductal cell layers toward endocrine cell phenotypes that synthesize and release proinsulin and subsequently insulin. HGF seems to provide a more efficient differentiation.