Hepatocyte growth factor (HGF) has been suggested to be a potent regulator of -cell function and proliferation. The purpose of this study was to investigate whether HGF could regulate the proliferation and differentiation of islet-derived epithelial monolayers into insulin-producing cells. We have generated islet-derived epithelial monolayers that are enriched with cells expressing c-Kit, a tyrosine kinase receptor and putative marker, from isolated postnatal rat islets. Monolayers were cultured on type I collagen gel and treated in defined differentiation medium with or without HGF (50 ng/ml) for 7 days. Subsequently, the expression of transcription factors and pancreatic endocrine cell markers as well as c-Kit expression were compared between the HGF (HGF + ), no HGF treatment (HGF ) and monolayers without differentiation medium (control) groups, using immunocytochemical and RT-PCR approaches. We observed that the number of c-Kit-, glucose transport type 2 (Glut2)-and the transcription factor pancreatic duodenal homeobox-1 (PDX-1)-expressing cells were significantly increased in the HGF + group. The expression of insulin at the mRNA and protein level was also increased in this treatment group with a 1·7-fold increase in basal insulin release and a 2·3-fold increase in insulin content in comparison with the HGF group. A high proliferative capacity was also found in the HGF + group. Co-localization of insulin and PDX-1 or Glut2 was revealed frequently in cells treated with HGF + with occasional co-staining of c-Kit and insulin observed. This study showed that HGF can activate the proliferation and differentiation of islet-derived epithelial monolayer into insulin-producing cells. However, no formation of islet-like clusters was observed. Taken together, this study implies that HGF mediates differentiation of immature cell types into insulin-expressing cells; however, HGF supplementation alone is insufficient in restoring full -cell function.