Recognizing that uterine stromal cells regulate several uterine epithelial cell function(s), the current study was undertaken to more fully define cell-cell communication in the uterus and to examine the role of uterine stromal cells in regulating epithelial cell monolayer integrity and cytokine release. Uterine epithelial and stromal cells from adult intact mice were isolated and cultured separately on cell culture inserts and/or in culture plates. Epithelial cells, which reach confluence as indicated by high transepithelial resistance (TER > 1000 ohms/well), preferentially release transforming growth factor-beta (TGFβ) into the basolateral chamber (≈70% > apical) and tumor necrosis factor-alpha (TNFα) into the apical compartment (≈30% > basolateral). When epithelial cells on cell culture inserts were transferred to plates containing stromal cells, coculture for 24–48 h increased epithelial cell TER (≈70% higher than control) and decreased TNFα release into both the apical and basolateral chambers (≈30%–50%). In contrast, TGFβ release was not affected by the presence of stromal cells. In other studies, the effects of stromal cells on epithelial cell TER and TNFα release persisted for 5–7 days following the removal of stromal cells and were also seen in coculture studies in which conditioned stromal media (CSM) was placed in the basolateral chamber. These studies indicate that uterine stromal cells produce a soluble factor(s) that regulates epithelial cell TER and release of TNFα without effecting TGFβ release. These results suggest that uterine stromal cells communicate with epithelial cells via a soluble factor(s) to maintain uterine integrity and epithelial secretory function.