Estrogens increase secretion of cervical mucus in females. The objective of this research was to study the mechanisms of estrogen action. The experimental models were human CaSki (endocervical) and hECE (ectocervical) epithelial cells cultured on filters. Incubation in steroid-free medium increased transepithelial electrical resistance (R_TE) and decreased epithelial permeability to the cell-impermeant acid pyranine. Estrogen treatment reversed the effects, indicating estrogen decreases epithelial paracellular resistance. The estrogen effect was time and dose related (EC₅₀ ~1 nM) and specific (estradiol = diethylstilbestrol > estrone, estriol; no effect by progesterone, testosterone, or cortisol) and was blocked by progesterone, tamoxifen, and ICI-182780 (an estrogen receptor antagonist). Estrogen treatment did not modulate dilution potential or changes in R_TE in response to diC8 or to low extracellular Ca²⁺ (modulators of tight junctional resistance). In contrast, estrogen augmented decreases in R_TE in response to hydrostatic and hypertonic gradients [modulators of resistance of lateral intercellular space (R_LIS)], suggesting estrogen decreases R_LIS. Estrogen decreased cervical cell size, shortened response time relative to changes in cell size after hypertonic challenge, and augmented the decrease in cell size in response to hypertonic and hydrostatic gradients. Lowering luminal NaCl had no significant effect on R_TE, and the Cl channel blocker diphenylamine-2-carboxylate attenuated the hypertonicity-induced decrease in cell size to the same degree in control and estrogen-treated cells, suggesting estrogen effects on permeability and cell size are not mediated by modulating Na⁺ or Cl transport. In contrast, estrogen increased cellular G-actin levels, suggesting estrogens shift actin steady-state toward G-actin and the cervical cell cytoskeleton toward a more flexible structure. We suggest that the mechanism by which estrogens decrease R_LIS and increase permeability is by fragmenting the cytoskeleton and facilitating deformability and decreases in cervical cell size.