Underlying stromal cells are essential for the normal development of epithelial cells (ECs) at mucosal surfaces. Recent studies from our laboratory have shown that uterine stromal cells regulate EC integrity, measured as transepithelial resistance (TER) as well as tumor necrosis factor (TNF) α α secretion by ECs in culture. Using stromal cells in coculture with polarized ECs grown on inserts, we found that stromal cells produce soluble mediators that increase TER and decrease TNFα secretion. The purpose of the present study was to identify the mechanisms whereby stromal cells exert their effects on uterine epithelium. We report that hepatocyte growth factor (HGF), a known mesenchymal growth factor that mediates EC proliferation, increases TER but, at the same time, decreases apical TNFα release. When ECs and/or stromal cells were incubated with anti-HGF or anti-HGF receptor (HGFR) antibody before HGF, the effects of HGF were blocked. These findings indicate that ECs express the HGFR at their basolateral surfaces and that HGFR mediates the effects of HGF on TER and TNFα. Neutralization of stromal cell secretions with antibodies for HGF and HGFR demonstrate that stromal-derived HGF is the mediator of EC TER. In contrast, neither anti-HGF antibody nor HGFR antibody had any effect on stromal cell-induced decreases in TNFα secretion. From these results, we conclude that stromal cell regulation of EC TER is mediated through the secretion of stromal HGF. Furthermore, because neutralization of stromal media failed to affect TNFα secretion, these findings suggest that other growth factors, in addition to HGF, affect EC cytokine production.