This project examined the regulation of hepatocyte growth factor (HGF) and stromal-derived factor 1 (SDF-1) secretion by primary cultures of human stromal fibroblasts of the uterus, cervix, and ectocervix. HGF is secreted constitutively in confluent monolayers of uterine, cervical, and ectocervical stromal fibroblasts. HGF secretion by uterine stromal fibroblasts continued beyond eight days of culture. Uterine stromal fibroblasts secrete increased HGF above constitutive levels in response to physiological concentrations of estradiol; maximal levels of secretion were reached by day four. Cell counts of uterine stromal fibroblasts exposed to media control or estradiol were not significantly different, so cell proliferation does not account for the increase in HGF secretion. In contrast, primary human cervical and ectocervical stromal fibroblasts were unresponsive to estradiol. This underscores the importance of uterine stromal fibroblasts in regulating uterine epithelial cell proliferation and function in response to estradiol. Uterine stromal fibroblasts increased HGF secretion in response to Toll-like receptor 3 (TLR 3) stimulation via Polyinosinic:polycytidylic acid (Poly (I:C)), a synthetic mimic of viral dsRNA. Poly (I:C)-induced HGF secretion was significantly higher than that induced by estradiol treatment, and treatment with estradiol plus Poly (I:C) had an additive effect on HGF secretion. Primary human cervical stromal fibroblasts from some donors increased HGF secretion in response to Poly (I:C), but estradiol did not modulate HGF secretion when cervical stromal fibroblasts were co-treated with estradiol plus Poly (I:C). Treatment of ectocervical stromal fibroblasts with Poly (I:C) resulted in increased HGF secretion, and again estradiol had no effect on Poly (I:C)-induced increases of HGF secretion when cells were co-treated with estradiol and Poly (I:C). In a preliminary experiment, TLR 3 mRNA was identified in uterine, cervical, and ectocervical stromal fibroblasts by RT-PCR. Stromal fibroblasts of the uterus, cervix, and ectocervix constitutively produce stromal-derived factor 1 (SDF-1), a paracrine molecule with similar function to HGF. However, the effects of estradiol and Poly (I:C) on SDF-1 secretion varied among donors; a much larger donor population will need to be studied to determine the effects of estradiol and Poly (I:C) on SDF-1 secretion by primary human uterine, cervical, and ectocervical stromal fibroblasts.