With increasing age the human skeleton decreases in density, thereby compromising its load-bearing capacity. Mechanical loading activates bone formation, but an age-dependent decrease in skeletal mechanoresponsiveness has been described in rats. In this paper we examine whether age-related bone loss is reflected by a decrease in the mechanosensitivity of isolated bone cells from human donors. Bone cell cultures were obtained from 39 donors (males and females) between 7 and 85 years of age. Cultures were challenged with 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) or mechanically stressed by treatment with pulsating fluid flow (PFF; 0.7 +/- 0.03 Pa at 5 Hz for 1 h). The growth capacity of the bone-derived cell population almost halved between 7 and 85 years of age. Basal alkaline phosphatase activity of the cells increased with donor age, while the response to 1,25(OH)2D3, measured as stimulated osteocalcin production, decreased with age. Together this suggests that the cell cultures from older donors represented a more mature, slower-growing cell population than the cultures from young donors. All cell cultures responded to mechanical stress with enhanced release of prostaglandin E2 (PGE2) and I2 (PGI2). The magnitude of the response was positively correlated with donor age, cell cultures from older donors showing a higher response than cultures from younger donors. There was also a positive correlation between time to reach confluency and mechanosensitivity, i.e., the PGE2 response to PFF treatment was higher in bone cell cultures with a slower growth rate. We conclude that bone cell cultures from older donors have a lower proliferative capacity and a higher degree of osteoblastic maturation than younger donors. The higher degree of osteoblastic maturation explains the higher response of the cultures to mechanical stress, in line with earlier studies on chicken bone cells. This study found no evidence for loss of mechanosensitivity with donor age. The reduced growth capacity might, however, be a factor in age-related bone loss.