Cells within the normal human colonic epithelium undergo a dynamic cycle of growth, differentiation and death. The organotypic culture system of human fetal colonic epithelial cells seeded on top of collagen gels with embedded colonic fibroblasts allowed prolonged culture of the colonic epithelial cells (Kalabis et al). Herein, we have evaluated the role of Endothelin-3 (ET3) and both cognate endothelin receptors (ETRA, ETRB) for human colonic epithelial cell growth and survival. ET3 was produced continuously by the fibroblasts as a result of adenovirus-mediated gene transfer. The presence and function of the endothelin receptors (ETRs) in epithelial cells was evaluated by 3H-thymidine incorporation using primary epithelial cells in monoculture and by immunohistochemistry on human fetal and adult paraffin embedded tissues. In organotypic culture, ET3 increased the number of goblet cells but not of enteroendocrine cells. The increase in goblet cells was caused by prolonged cell survival and differentiation. The inhibition of both ETRA and ETRB decreased significantly the number of goblet cells and proliferation in epithelial cells, while the number of enteroendocrine cells remained unchanged. ET3 induced activation of IB and MAPK in the epithelial cells suggesting these signaling pathways mediate its pro-proliferation and pro-survival activities. Our results demonstrate that ET3 is involved in regulating human colonic epithelial cell proliferation and survival, particularly for goblet cells, and may be an important component of colonic homeostasis.