Using a parallel-plate flow chamber and fura-2 fluorescence microscopy, intracellular calcium was measured cell by cell in preconfluent primary culture rat calvarial bone cells to 18, 35, and 70 dynes/cm² of fluid-induced shear stress. A heterogeneous response with respect to peak amplitude and latency was observed for the culture, with an overriding dose-dependent relationship between the mean peak amplitude of response and shearstress magnitude. A dose dependence was observed between the number of responsive cells (responding >50% over basal levels) and shear-stress magnitude. Not all cells could be restimulated by repeated exposure to flow. The observed cell response appears to be independent of whether cells are clustered together or isolated. Substratum stretch, hydrostatic pressure, and fluid shear stress have been shown in the literature to increase inositol phosphate (IP,3) in bone cells, with IP₃ causing the release of calcium from intracellular stores such as the endoplasmic reticulum. Therefore, a 6-fold inhibitory effect observed when calcium release from stores was blocked with 8-(n,N-diethylamino)octyl 1-3,4,5-atrimethoxybenzoate hydrochloride implicates an IP₃ biochemical pathway mediating the fluid flow response in bone cells.