In this study we provide the first evidence that human non-osteoblastic cells secrete the bone-specific protein osteocalcin. We show that the myeloma cell line NCI-H929 constitutively produces low amounts of osteocalcin. Furthermore, we demonstrate that this production is strongly enhanced after 1,25(OH)2D3 stimulation. An amplification or a deregulation of the osteocalcin gene could be responsible for the observed phenomenon because NCI-H929 has the unusual characteristic of having at least three chromosomes 1, where the osteocalcin gene was mapped (1q25-q31). It is of interest to note that the patient in whom the NCI-H929 cell line originated never developed osteolytic lesions despite extensive disease apparent at autopsy. Although lytic bone lesions with low osteocalcin serum levels are a common feature of multiple myeloma (MM), a small subset of patients does not develop lytic bone lesions or even demonstrate osteosclerotic MM. This occurrence is associated with high osteocalcin serum levels of unknown origin. In this context, osteocalcin production by immortalized tumor lacking any osteolytic potential could be relevant to the pathophysiology of this particular type of MM.