Age-related bone loss may be a consequence of a lack of osteoblastic formation and/or function. In vitro, the osteoblastic response to 1,25(OH)2D3, an important regulator of osteoblastic function, appears to depend on the stage of osteoblastic maturation. In this study, we examined the response to 1,25(OH)2D3 of C-terminal type I procollagen (PICP), alkaline phosphatase (ALP), and osteocalcin (OC) secretion in primary cultures of osteoblastic cells from human trabecular bone (hOB). Forty-four bone samples were obtained from subjects undergoing knee arthroplastia, 20 aged 50-70 (64 +/- 5), and 24 >70 (73 +/- 2) years. Another 33 bone samples were obtained from subjects undergoing hip arthroplastia, 21 were aged 50-70 (64 +/- 4) and 12 >70 (75 +/- 5) years. Pooling knee and hip hOB cell cultures, we found that PICP secretion decreased after 1,25(OH)2D3 in hOB cells from the older group (>70 years). Treatment with 1,25(OH)2D3 increased ALP secretion in these cells only in the younger group (50-70 years), whereas it increased OC secretion in hOB cells in both age groups. By pooling hOB cell cultures from both age groups we found that knee hOB cells increased OC secretion, and decreased PICP secretion, after 1,25(OH)2D3. This metabolite also increased OC secretion in hip hOB cells. Considering the influence of donor age at the same skeletal site, 1,25(OH)2D3 was found to stimulate ALP secretion only in knee hOB cells in the younger group. In contrast, this metabolite decreased ALP secretion in hip hOB cells in the older group. PICP secretion decreased after 1,25(OH)2D3 only in hOB cells in the older group, at both skeletal sites. In age-matched cultures, OC secretion was lower in hip hOB cells compared with those from the knee in the older group, but was similar in these cell cultures from both skeletal sites in the younger group. OC secretion after 1,25(OH)2D3 stimulation did not show age differences in knee hOB cells, but was lower in hip hOB in the older group. In summary, our results demonstrate that the response of various osteoblastic markers to 1,25(OH)2D3 in primary cultures of hOB cells depends on the donor age and skeletal site of origin.