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 Ca/sup 2+/ ([Ca/sup 2+/]) and Ca/sup 2+/ channels in these responses were examined using agents that alter [Ca/sup 2+/]/sub i/, release and Ca/sup 2+/ entry. The intracellular Ca/sup 2+/ chelator, BAPTA, abolished flow-induced ASFF and gene expression, but Ca/sup 2+/ channel blockers, nifedipine and gadolinium, failed to inhibit these responses. Thapsigargin, which empties [Ca/sup 2+/]/sub i/, stores, and U73122, a phospholipase C inhibitor, completely suppressed flow-induced ASFF and c-fos/COX-2 expression. These data indicate that IP/sub 3/-mediated [Ca/sup 2+/]/sub i/ release is essential for these flow induced responses in osteoblasts. However, the authors found that localization of Ca/sup 2+/ channels to the membrane was required for the [Ca/sup 2+/]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.