The content and affinity of calcitriol receptors were analyzed in cultured osteoblasts from normal and hypophosphatemic mice. Hypertonic cell extracts were prepared by sonication followed by centrifugation at 200,000 g × 30 min. Analysis, at saturating levels of labeled 1,25(OH)₂D₃, revealed that binding of the hormone was dependent on the density of the cells plated and on the length of time in culture. It reached a maximum at 5 days of culture when 1.0 × 10⁶ cells were plated. Under those conditions the binding capacity of Hyp osteoblasts was 6306 ± 1267 sites/ng protein (mean ± SEM) not different from N cells (7594 ± 1713). The dissociation constant (Kd) was 18.3 ± 5.4 and 20.0 ± 5.7 pM for mutant and normal mouse osteoblasts respectively (NS). In both genotypes, a single peak for specific binding, migrating at approximately 3.0–3.5 S was observed by sucrose gradient centrifugation. 25-hydroxycholecalciferol-24-hydroxylase (24-OHase) was induced at 1 and 10 nM 1,25(OH)₂D₃ in a dose-dependent fashion. However, the induction was higher in mutant than in normal cells when the medium contained 1 mM and 2 mM phosphate salts. The difference vanished when cells were incubated in the presence of 3 and 4 mM phosphate salts. The effect of calcitriol on cultured osteoblasts was also analyzed in terms of collagen synthesis and alkaline phosphatase activity. In the range of 10⁻¹⁰ M to 10⁻⁷ M, 1,25(OH)₂D₃ was found to inhibit collagen synthesis in a dose-dependent fashion. At physiological levels, 1,25(OH)₂D₃ (10⁻¹¹ M−10⁻¹⁰ M), stimulated alkaline phosphatase activity. However at higher concentrations (10⁻⁸ M−10⁻⁷ M) it provoked an inhibition. The data show that there is no alteration in the receptor binding capacity and the receptor affinity for calcitriol in Hyp mouse osteoblasts. They also show that at 1 mM phosphate stimulation of alkaline phosphatase and inhibition of collagen synthesis by 1,25(OH)₂D₃ were similar in both cell strains and that the magnitude of the induction of 24-OHase is influenced by the extracellular phosphate concentration in hypophosphatemic but not normal cells.