The transient receptor vanilloid-4 (TRPV4) is a mechanosensitive, swell-activated cation channel that is abundant in the renal distal tubules. Immunolocalization studies, however, present conflicting data as to whether TRPV4 is expressed along the apical and/or basolateral membranes. To disclose the role of TRPV4 in flow-dependent K⁺ secretion in distal tubules in vivo, urinary K⁺ excretion and net transports of K⁺ and Na⁺ in the cortical collecting duct (CCD) were measured with an in vitro microperfusion technique in TRPV4^+/+ and TRPV4^−/− mice. Both net K⁺ secretion and Na⁺ reabsorption were flow dependently increased in the CCDs isolated from TRPV4^+/+mice, which were significantly enhanced by a luminal application of 50 μM 4α-phorbol-12,13-didecanoate (4αPDD), an agonist of TRPV4. No flow dependence of net K⁺ and Na⁺ transports or effects of 4αPDD on CCDs were observed in TRPV4^−/− mice. A basolateral application of 4αPDD had little effect on these ion transports in the TRPV4^+/+ CCDs, while the luminal application did. Urinary K⁺ excretion was significantly smaller in TRPV4^−/− than in TRPV4^+/+ mice when urine production was stimulated by a venous application of furosemide. These observations suggested an essential role of the TRPV4 channels in the luminal or basolateral membrane as flow sensors in the mechanism underlying the flow-dependent K⁺ secretion in mouse CCDs.