Second-messenger systems have been implicated to transmit mechanical stimulation into cellular signals; however, there is no information on how mechanical stimulation is affected by such systemic factors as parathyroid hormone (PTH). Regulation of adenylyl cyclase and phosphatidylinositol pathways in rat dentoalveolar bone cells by mechanical strain and PTH was investigated. Two different cell populations were isolated after sequential enzyme digestions from dentoalveolar bone (group I and group II) to study potential differences in response. Mechanical strain was applied with 20 kPa of vacuum intermittently at 0.05 Hz for periods of 0.5, 1, 5, 10, and 30 minutes and 1, 3, and 7 days using the Flexercell system. Levels of cAMP, measured by RIA, and levels of inositol 1,4,5-triphosphate (IP3) and protein kinase C activity (PKC), measured by assay systems, increased with mechanical strain. When PTH was added to the cells, there was a significant increase in levels of all the intracellular signals, which appeared to potentiate the response to mechanical strain. IP3 levels (0.5 minute) peaked before those of PKC activity (5 minutes), which in turn peaked before those of cAMP (10 minutes). Group II cells showed higher levels of cAMP and IP3 than the group I cells. This suggests that the former may ultimately play the predominant roles in skeletal remodeling in response to strain. Immunolocalization of the cytoskeleton proteins vimentin and α-actinin, focal contact protein vinculin, and PKC showed a marked difference between strained and nonstrained cells. However, the addition of PTH did not cause any significant effect in cytoskeleton reorganization. Staining of PKC and vimentin, α-actinin, and vinculin suggests that PKC participates actively in the transduction of mechanical signals to the cell through focal adhesions and the cytoskeleton, although only PKC seemed to change with short time periods of strain. In conclusion, dentoalveolar osteoblasts responded to mechanical strain initially through increases in levels of IP3, PKC activity, and later cAMP, and this response was potentiated when PTH was applied together with mechanical strain.