C57BL/6J Hyp/Y male mice, and X-linked hypophosphatemic human males are dwarfed and have rickets responsive to orthophosphate (Pi). Serum Pi in mature Hyp/Y mice is low (4.6±0.9mg/dl) vs normal +/Y male littermates (7.6±1.Omg/dl, p<0.001). Serum calcium is low normal in Hyp/Y. The renal “excretion index” (mg Pi in urine/mg creat.)/(mg Pi/ml serum) is 103±25 vs 65±16 in +/Y mice (p<0.01). Endogenous kidney cortex Pi is 314μg/g wet wt. in Hyp/Y despite hypophosphatemia, & 307μg/g in +/Y mice. Concentration-dependent uptake of 32Pi into organic and inorganic pools in renal cortex slices is normal in Hyp/Y kidney, under initial-rate and steady-state conditions. The mutant gene product in Hyp/Y is apparently limited to the luminal surface in the kidney tubule since net reabsorption in vivo (luminal membrane) is depressed, while in vitro uptake by slices (basilar membrane) is normal. Preliminary evidence for a luminal membrane defect is also present in Hyp/Y intestine. These findings offer the first in vitro evidence to support the hypothesis that the XLH gene affects Pi luminal back-flux during transepithelial absorption.