Extracellular ATP is an autocrine/paracrine factor that regulates renal function. TRPV4 is a cation channel that mediates release of autocrine/paracrine factors by acting as an osmosensor. The renal medulla, and therefore the thick ascending limb, is exposed to osmotic stress. We hypothesize that reduced osmolality stimulates ATP release from the thick ascending limb via TRPV4 activation. We measured ATP release by medullary thick ascending limb suspensions after reducing bath osmolality from 350 to 323 mOsm/KgH₂O, using the luciferin-luciferase assay. Decreasing osmolality stimulated ATP release compared to control (38.9±7.2 vs. 2.4±1.0 pmol/mg protein; n=6, p<0.01). To examine the role of TRPV4, we used 1) Ca-free solutions 2) a TRPV4 inhibitor 3) siRNA against TRPV4 and 4) a TRPV4 activator. Removal of Ca completely blocked osmolality-induced ATP release (42.2±5.9 vs. 2.6±1.5 pmol/mg protein; n=6, p<0.01). In the presence of the TRPV4-selective inhibitor ruthenium red, osmolality-induced ATP release was blocked by 73% (56.4±19.9 vs. 8.8±2.3 pmol/mg protein; n=6; p<0.03). In vivo treatment of thick ascending limbs with siRNA against TRPV4 decreased osmolality-induced ATP release by 62% (31.5±3.4 vs. 12.4±1.1 pmol/mg protein; n=7; p<0.01), while reducing TRPV4 expression by 74% compared to the non-treated kidney. Treatment with scrambled siRNA did not affect TRPV4 expression and/or osmolality-induced ATP release. Finally, in the absence of changes in osmolality, the specific TRPV4 agonist 4-PDD increased ATP release (3.6±0.9 vs. 25.4±7.4 pmol/mg protein; n=6; p<0.04). We concluded that decreases in osmolality stimulate ATP release by thick ascending limbs and this effect is mediated by TRPV4 activation.