Vascular smooth muscle cells (VSM) are normally exposed to transmural fluid flow shear stresses, and after vascular injury, blood flow shear stresses are imposed upon them. Since Ca2+ is a ubiquitous intracellular signaling molecule, we examined the effects of fluid flow on intracellular Ca2+ concentration in rat aortic smooth muscle cells to assess VSM responsiveness to shear stress. Cells loaded with fura 2 were exposed to steady flow shear stress levels of 0.5-10.0 dyn/cm2 in a parallel-plate flow chamber. The percentage of cells displaying a rise in cytosolic Ca2+ ion concentration ([Ca2+]i) increased in response to increasing flow, but there was no effect of flow on the ([Ca2+]i) amplitude of responding cells. Addition of Gd3+ (10 microM) or thapsigargin (50 nM) significantly reduced the percentage of cells responding and the response amplitude, suggesting that influx of Ca2+ through ion channels and release from intracellular stores contribute to the rise in ([Ca2+]i) in response to flow. The addition of nifedipine (1 or 10 microM) or ryanodine (10 microM) also significantly reduced the response amplitude, further defining the role of ion channels and intracellular stores in the Ca2+ response.