The objective of this study was to assess estrogen-dependent cellular mechanisms that could contribute to the acid pH of the vaginal lumen. Cultures of normal human cervical-vaginal epithelial cells (hECE) and endocervical cells were grown on filters, and acidification of the extracellular solutions on the luminal (L-pHo) and contra-luminal (CL-pHo) sides was measured. hECE and endocervical cells decreased CL-pHo from 7.40 to 7.25 within 20-30 min of incubation in basic salt solution. Endocervical cells produced a similar decrease also in L-pHo. In contrast, hECE cells acidified L-pHo down to pH 7.05 when grown as monoculture, and down to pH 6.05 when grown in co-culture with Human Cervical Fibroblasts. This enhanced acid secretion into the luminal compartment was estrogen dependent, since removal of endogenous steroid hormones attenuated the effect while treatment with 17-estradiol restored it. The 17-estradiol effect was dose-dependent (EC₅₀ 0.5 nM) and could be mimicked by diethylstilbestrol and in part by estrone and tamoxifen. Pre-incubation with ICI-182780, but not with progesterone, blocked the estrogen effect. Pre-incubation of cells with the V-ATPase blocker bafilomycin A₁, when administered to the luminal solution, attenuated the baseline and estrogen-dependent acid secretion into the luminal solution. Treatment with EGTA, to abrogate the tight junctional resistance, blocked the decrease in L-pHo and stimulated a decrease in CL-pHo, indicating that the tight junctions are necessary for maintaining luminal acidification. We conclude that: 1. Vaginal-ectocervical cells acidify the luminal canal by a mechanism of active proton secretion, driven in part by V-H⁺-ATPase located in the apical plasma membrane; 2. the baseline active net proton secretion occurs constitutively throughout life and that this acidification is upregulated by estrogen.