Fibroblast growth factor 23 (FGF23) is a phosphaturic factor that suppresses both sodium-dependent phosphate transport and production of 1,25-dihydroxyvitamin D [1,25(OH)(2)D] in the proximal tubule. In vitro studies suggest that FGFR3 is the physiologically relevant receptor for FGF23 in the kidney, but this has not been established in vivo. Here, immunohistochemical analysis of the mouse kidney revealed that the proximal tubule expresses FGF receptor 3 (FGFR3) but not FGFR1, FGFR2, or FGFR4. Compared with wild-type mice, Hyp mice, which have elevated circulating levels of FGF23, exhibited low levels of serum phosphate and 1,25(OH)(2)D, reduced expression of the sodium-dependent phosphate transporter NPT2a in the proximal tubules, and low bone mineral density as a result of osteomalacia. In contrast, neither the serum phosphate nor 1,25(OH)(2)D levels were altered in FGFR3-null mice. For examination of the role of FGFR3 in mediating the effects of FGF23, Hyp mice were crossed with FGFR3-null mice; interestingly, this failed to correct the aforementioned metabolic abnormalities of Hyp mice. Ablation of FGFR4 also failed to correct hypophosphatemia in Hyp mice. Because the ablation of neither FGFR3 nor FGFR4 inhibited the renal effects of excess FGF23, the kidney localization of FGFR1 was investigated. FGFR1 co-localized with Klotho, the co-factor required for FGF23-dependent FGFR activation, in the distal tubule. In summary, neither FGFR3 nor FGFR4 is the principal mediator of FGF23 effects in the proximal tubule, and co-localization of FGFR1 and Klotho suggests that the distal tubule may be an effector site of FGF23.