Many vertebrate cells react to hypotonic conditions with swelling, followed by an active downregulation of the cell volume; a progress called regulatory volume decrease (RVD). While the actual process of volume decrease by loss of osmotically active molecules like K(+) and Cl(-), followed by water efflux has been extensively investigated, the signal for activation of RVD still remains obscure. Studies with different cell lines demonstrated a participation of the cation channel transient receptor potential vanilloid 4 (TRPV4) as well as the actin cytoskeleton in volume regulation. Therefore, we analyzed putative links between TRPV4 and F-actin in RVD in HaCaT keratinocytes and CHO cells. Laser scanning microscopy studies revealed a distinct colocalization of TRPV4 and actin in highly dynamic membrane structures, such as microvilli, filopodia and lamellipodia edges. After treatment of cells with the actin-destabilizing reagent latrunculin A, TRPV4 and F-actin no longer colocalized within the membrane. In accordance with these data, close interaction between TRPV4 and F-actin was revealed by FRAP and FRET studies. For functional analysis, CHO cells that endogenously do not express TRPV4, were transfected with recombinant TRPV4, which rendered them RVD-competent. Treatment with latrunculin A abolished both, RVD and the accompanying rise of [Ca(2+)](i) after hypotonic stress in TRPV4-transfected CHO cells. Taken together, our data demonstrate a functional interaction between TRPV4 and F-actin in sensing hypotonicity and the onset of RVD.