Alterations in osteoblast metabolism are involved in the pathogenesis of typical subchondral bone changes in osteoarthritis (OA). Osteocalcin is a specific bone protein, synthesised by the osteoblasts, which can be considered a marker of metabolic activity of these cells. In this study we correlated osteocalcin production from human osteoblasts isolated from healthy and osteoarthritic subjects to the degree of cartilage damage, before and after stimulation with 1,25(OH)2-vitamin D3, the active metabolite of vitamin D3. We isolated human osteoblasts from cancellous bone of healthy subjects and from subchondral bone of osteoarthritic subjects and considered the osteoblasts corresponding to different degrees of cartilage damage as different cell populations. We determined the osteocalcin production in normal and osteoarthritic osteoblasts from maximal and minimal cartilage damage areas both under basal conditions and after vitamin D3 stimulation. Compared to normal osteoblasts, under basal conditions osteocalcin production is significantly greater in osteoarthritic osteoblasts, corresponding both to maximal and minimal damage joint areas. No differences were observed between osteoblasts from maximal and minimal damage areas. The response of osteoblasts to vitamin D3 stimulation appeared to be proportional to the degree of joint damage, as the vitamin D3-induced increase in osteocalcin is proportionally greater in maximally damaged osteoblasts compared to minimally damaged ones. Thus, after vitamin D3 stimulation, a significant increase in osteocalcin production by maximally damaged osteoblasts compared to the minimally damaged ones was observed. This study confirms abnormal osteoarthritic osteoblast behaviour and indicates that osteoblasts from different areas of the same affected joint may be metabolically different, supporting the hypothesis that subchondral osteoblasts may play an essential role in the pathogenesis of OA.