Hypophosphatemic vitamin D-resistant rickets is characterized by impaired renal reabsorption of Pi. The underlying mechanism of this abnormality remains unknown. Because the osteoblast is likely a target for the HYP mutation, we investigated the Pi transport activity in osteoblasts isolated from the murine homologue for the human disease, the Hyp mouse. Kinetic analysis of sodium-dependent Pi uptake in quiescent normal and Hyp osteoblasts indicated no significant differences in apparent maximal capacity (Vmax) and apparent affinity (Km) of the carrier for Pi. In rapidly growing cells, higher levels of Pi uptake were observed in mutants cells associated with a 1.4- to 1.7-fold increase in Vmax and no change in Km for Pi. This increase in Pi uptake seemed related to changes in the sodium electrochemical gradient inasmuch as a similar increase was observed in alanine uptake. The adaptive response of sodium-dependent Pi transport to Pi deprivation was not altered in mutant cells relative to normal cells. To test whether the expression of a Pi transport defect was dependent on a humoral factor for its expression, we evaluated the activity of the serum from Hyp mice on Pi transport in osteoblasts from both genotypes. No difference in activity was observed between sera from normal and mutant mice. In summary, cultured osteoblasts derived from Hyp mice did not express impaired sodium-dependent Pi transport when compared with cells from normal mice.