We have shown earlier that mechanical stimulation by intermittent hydrostatic compression (IHC) promotes alkaline phosphatase and procollagen type I gene expression in calvarial bone cells. The bone matrix glycoprotein osteopontin (OPN) is considered to be important in bone matrix metabolism and cell‐matrix interactions, but its role is unknown. Here we examined the effects of IHC (13 kPa) on OPN mRNA expression and synthesis in primary calvarial cell cultures and the osteoblast‐like cell line MC3T3‐E1. OPN mRNA expression declined during control culture of primary calvarial cells, but not MC3T3‐E1 cells. IHC upregulated OPN mRNA expression in late released osteoblastic cell cultures, but not in early released osteoprogenitor‐like cells. Also, in both proliferating and differentiating MC3T3‐E1 cells, OPN mRNA expression and synthesis were enhanced by IHC, differentiating cells being more responsive than proliferating cells. These results suggest a role for OPN in the reaction of bone cells to mechanical stimuli. The severe loss of OPN expression in primary bone cells cultured without mechanical stimulation suggests that disuse conditions down‐regulate the differentiated osteoblastic phenotype. J. Cell. Physiol. 170:174–181, 1997. © 1997 Wiley‐Liss, Inc.