The thick ascending limb (TAL) reabsorbs 30 % of the filtered NaCl. Thus it plays a pivotal role in the regulation of salt homeostasis. Luminal flow enhances both shear stress and cellular stretch. The mechanosensitive Transient Receptor Potential Vanilloid 4 (TRPV4) channel mediates increases in intracellular calcium (Cai) in response to shear stress and cellular stretch in many cells. However, it is not known whether shear stress and/or cellular stretch mediate flow‐induced Cai increases in TALs. We hypothesized that shear stress and cellular stretch mediate flow‐induced Cai increases in TALs. We measured Cai using the ratiometric Ca‐sensitive dye Fura‐2 in isolated and perfused rat TALs. Increasing luminal flow increased Cai from 54 ± 7 to 410 ± 116 nM, n=6; p<0.03. The TRPV4 antagonist RN 1734 (50 uM) blocked this effect (from 29 ± 7 to 80 ± 11 nM, n=5). To assess the contributions of shear stress and stretch we measured Cai at a location close to the perfusion pipet where shear stress and stretch would be high and at a distal site at the end of the tubule where shear stress would be the same as the proximal site but stretch would be greatly reduced. Luminal flow increased Ca by 0.75 ± 0.11 ratio units (RU) in the proximal region. In the distal region, flow increased Ca by 0.45 ± 0.10 RU (n=5), 40% less than in the proximal region (p<0.002). The general TRPV4 antagonist ruthenium red blocked the increase in both regions. We conclude that: 1) flow increases intracellular Ca via TRPV4 in TALs; and 2) shear stress mediates most of the effect but cellular stretch also likely contributes.Grant Funding Source: Supported by NHLBI of NIH to J.G (HL 070985; HL 090550; HL 028982). AHA to P.C (13POST16280001)