Fluid flow induces increased c-fos and COX-2 expression in MC3T3-E1 cells that is dependent on flow-induced actin stress fiber formation (ASFF). The roles of intracellular Ca2+ ([Ca2+]i) and Ca2+ channels in these responses were examined using agents that alter [Ca2+]i release and Ca2+ entry. The intracellular Ca2+ chelator, BAPTA, abolished flow-induced ASFF and gene expression, but Ca2+ channel blockers, nifedipine and gadolinium, failed to inhibit these responses. Thapsigargin, which empties [Ca2+]i stores, and U73122, a phospholipase C inhibitor, completely suppressed flow-induced ASFF and c-fos/COX-2 expression. These data indicate that IP3-mediated [Ca2+]i release is essential for these flow induced responses in osteoblasts. However, we found that localization of Ca2+ channels to the membrane was required for the [Ca2+]i response to flow in MC3T3-E1 cells. Inhibition of these channels also reduced proliferation and increased alkaline phosphatase activity suggesting that these channels may be important to the differentiated state of osteoblastic cells.