Our previous studies suggested that increased osteoclast formation and activity in Paget's disease may be related in part to increased responsiveness of highly purified osteoclast precursors to 1,25‐dihydroxyvitamin D₃ [1,25‐(OH)₂D₃]. However, the basis for this enhanced sensitivity to 1,25‐(OH)₂D₃ is unclear. To address this question, we examined 24‐hydroxylase and 1,25‐(OH)₂D₃ receptor (VDR) messenger RNA (mRNA) expression during human osteoclast differentiation from normal subjects and patients with Paget's disease in response to 1,25‐(OH)₂D₃ as well as VDR content and affinity. Reverse‐transcription polymerase chain reaction (RT‐PCR) analysis of granulocyte–macrophage colony–forming unit (GM‐CFU), the earliest identifiable osteoclast precursor, derived from patients with Paget's disease demonstrated 24‐hydroxylase mRNA expression in response to 1,25‐(OH)₂D₃ was induced at concentrations of 1,25‐(OH)₂D₃ that were at least one log less than that required for normal GM‐CFU. VDR mRNA and VDR protein were detected in both immature and more differentiated osteoclast precursors, as well as in osteoclast‐like multinucleated cells (MNCs). However, VDR expression was lower in MNCs than the mononuclear precursor cells. Osteoclast precursors and MNCs from patients with Paget's disease had levels of VDR expression similar to those of normal subjects but showed increased VDR affinity for 1,25‐(OH)₂D₃. Because the effects of 1,25‐(OH)₂D₃ are in part mediated by induction of expression of RANK ligand on marrow stromal cells, which in turn stimulates osteoclast formation, we examined expression of RANK ligand mRNA by marrow stromal cell lines derived from patients with Paget's disease and normal subjects in response to 1,25‐(OH)₂D₃. RT‐PCR analysis showed no difference in sensitivity of marrow stromal cells to 1,25‐(OH)₂D₃ from normal subjects or patients with Paget's disease although the Paget's stromal cells expressed increased basal levels of RANK ligand mRNA. These results show that VDR protein is expressed in early and more differentiated osteoclast precursors, that expression levels of VDR decline with osteoclast differentiation, and that 1,25‐(OH)₂D₃ has direct effects on osteoclast precursors. The enhanced sensitivity to 1,25‐(OH)₂D₃ is an intrinsic property of osteoclast precursors from patients with Paget's disease that distinguishes them from normal osteoclast precursors. Furthermore, our results suggest that an increased affinity of VDR for 1,25‐(OH)₂D₃ may be responsible for the enhanced 1,25‐(OH)₂D₃ sensitivity of osteoclast precursors in patients with Paget's disease compared with normal subjects. (J Bone Miner Res 2000;15:228–236)