Medullary thick ascending limbs (mTAL) regulate Na balance and therefore blood pressure. We previously showed that cell swelling and luminal flow activates the mechanosensitive channel TRPV4 in mTAL. OBJECTIVE We hypothesized that TRPV4 mediates flow-induced increases in intracellular Ca (Cai) in rat mTALs. METHODS We performed ratiometric measurements of Cai in perfused mTALs. RESULTS Increasing luminal flow from 0 to 20 nL min(-1) caused Cai to peak 231 ± 29 nmol L(-1) above basal concentrations (n = 18). The general TRPV inhibitor ruthenium red at 15 and 50 μmol L(-1) reduced peak Cai by 41 ± 9 (P < 0.01; n = 5) and 77 ± 10% (P < 0.02; n = 6). The selective TRPV4 inhibitor RN1734 at 10 and 50 μmol L(-1) reduced peak Cai by 46 ± 11 (P < 0.01; n = 7) and 76 ± 5% (P < 0.02; n = 5) respectively. To specifically target TRPV4, mTALs were transduced with adenoviruses expressing TRPV4 small hairpin (sh) RNA. In non-transduced control mTALs, luminal flow generated a peak increase in Cai of 111 ± 21 nmol L(-1) (n = 8). In TRPV4shRNA-transduced mTALs, the Cai peak was reduced to 56 ± 8 nmol L(-1) (P < 0.03, n = 9). Removing extracellular Ca completely abolished flow-induced increases in Cai. Increasing luminal flow in the presence of hexokinase 20 (U mL(-1) ) to scavenge extracellular ATP did not modify significantly the increases in Cai induced by luminal flow. Finally, we studied the effect of the TRPV4 selective agonist GSK1016790A on Cai. In the absence of luminal flow, GSK1016790A (10 nmol L(-1) ) increased Cai from 60 ± 11 nmol L(-1) to 262 ± 71 nmol L(-1) (P < 0.05; n = 7). CONCLUSIONS We conclude that flow-induced increases in Cai are mediated primarily by TRPV4 in the rat mTAL.