The objective of the study was to understand how estrogen modulates the rigidity of the cytoskeleton in epithelial cells. Estrogen depletion decreased, and treatment with 17-estradiol increased deformability of cervical-vaginal epithelial cells. Estrogen also induced redistribution of non-muscle myosin-II-B (NMM-II-B); lesser interaction of NMM-II-B with actin; increased phosphorylation of NMM-II-B-heavy-chains at threonine and serine residues, and decreased filamentation of NMM-II-B in vitro. The effects of 17-estradiol were time- and dose-related, and could be mimicked by DES. The effects of estrogen were blocked by co-treatment with antisense oligonucleotide for the ER; and inhibited by ICI-182,780 and tamoxifen, by omission of EGF from the culture medium, and by co-treatments with the EGF receptor inhibitor AG1478, the ERK-MAPK inhibitor PD98059, the CK2 inhibitor DRB, the ROCK inhibitor Y-27632, and the non-specific phosphatase inhibitor okadaic acid. Co-administration of DRB plus okadaic acid blocked 17-estradiol effect. H-89 or LY294002 did not significantly affect estrogen effects. Treatment with estrogen increased activation of ERK1/2 and CK2 activity. These data suggest a novel pathway of estrogen regulation of the cytoskeleton in epithelial cells. The effect is mediated by ER and involves in part the EGF-EGFR and ERK-MAPK cascades as proximal signaling networks, and the CK2 and ROCK-regulated myosin heavy-chain phosphatase as terminal effectors. Augmented phosphorylation of NMM-II-B can block filamentation and induce disassociation of the myosin from the cortical actin, and disruption of the acto-myosin ring can increase cell deformability. This mechanism can explain estrogen regulation of paracellular permeability in cervical-vaginal epithelia in vivo.