A 97-kDa protein called valosin-containing protein (VCP) has been implicated in osteosarcoma metastasis and Paget's disease of bone, two conditions that complicate the course and outcome of orthopaedic surgery. High VCP gene expression is associated with high metastatic potential in osteosarcoma cells, while loss-of-function VCP mutations cause inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia (IBMPFD). VCP protein expression and regulation have not been examined in normal osteoblasts. The purpose of these studies was to characterize VCP protein expression in control and stressed untransformed osteoblasts. Proteins from confluent MC3T3-E1 mouse osteoblast-like cells were separated by 2D IEF/SDS-PAGE. An abundant spot with a M(r) of 94 kDa and a pI of 5.4 was identified as VCP by MALDI/ToF and peptide mass fingerprint analysis. High constitutive VCP protein expression in subconfluent and confluent resting and mildly physiologically stressed MC3T3-E1 cells was confirmed by Western blotting. When assessed by indirect immunofluorescence in fixed cells or Western blotting of subcellular fractions, VCP was more abundant in the cytoplasm than in the nucleus. Induction of mild physiological stress sufficient to stimulate the ubiquitin-proteasome pathway, which is partially dependent on VCP-mediated targeting of polyubiquitinylated substrates, did not affect steady-state VCP levels or distribution. Thus, VCP is a constitutively abundant protein in untransformed osteoblastic cells under all conditions tested. Such high levels of VCP protein expression in untransformed osteoblastic cells argue against a major causative role for it in metastasis, while the occurrence of Paget's disease in patients with missense VCP mutations supports a major role for VCP in normal osteoblast proliferation and regulation.