TRPV4 is a calcium-permeable channel activated by extracellular hypotonicity, polyunsaturated fatty acids, phorbol esters, and heat. We show that TRPV4 is localized in the basolateral membrane of the mouse mammary cell line HC11. Activation of TRPV4 caused an increase in the intracellular Ca(2+) concentration through influx of extracellular Ca(2+), triggering two independent chains of events: 1) a rapid increase in transcellular conductance through the activation of apical large conductance calcium-activated (BK) potassium channels that were blockable by paxilline; 2) a slow increase in paracellular permeability for small solutes. The latter effect was accompanied by a down-regulation of the tight junctional proteins claudin -1, -3, -4, -5, -7, and -8 and by dramatic changes in tight junction morphology, including frequent large breaks in the tight junction strands. This dual modulation of epithelial permeability after TRPV4 activation may be involved in regulating the tonicity across mammary gland epithelia. TRPV4 activation may also be responsible for exudation and edema formation during inflammation processes.-Reiter, B., Kraft, R., Günzel, D., Zeissig, S., Schulzke, J-D., Fromm, M., Harteneck, C. TRPV4-mediated regulation of epithelial permeability.