To explore lineage-dependent responses to mechanical stress in bone cells, newborn rat calvarial cells, exhibiting differential characteristics of osteoblastic and osteocytic cells, were compared in their immediate and late responses to stretching. Seven fractions of sequentially prepared cells were cultured on Matrigel to promote their differentiation. By cyclically stretching the flexible bottom of culture plates, cells were exposed to a physiological stress of approximately 4000 microstrain on Matrigel. Cells in fractions IV, V and VI exhibited striking responses; the levels of cAMP and insulin-like growth factor I, bone Gla protein, and mineral accumulation were significantly elevated in the stretched cells. Also, proliferation was significantly inhibited regardless of the presence of 10-6 M indomethacin. In contrast, osteoblasts in fraction III and osteocyte-like cells in fraction VII exhibited no significant response. Thus, these intermediate cells, very mature osteoblasts to young osteocytes, are likely to serve as a mechanosensor in bone, controlling the metabolic aspects of physical stress. We conclude that the responses of these young osteocytes to low level, physiological strain are transmitted in a manner different from the responses of osteoblasts to higher magnitude of strain in which PGE₂ induces cell proliferation, as reported by others.