The relationships between fluid shear stress, a physiologically relevant mechanical force in the circulatory system, and pinocytosis (fluid-phase endocytosis) were investigated in cultured bovine aortic endothelial cells using a specially designed apparatus. Continuous exposure to steady shear stresses (1-15 dyn/cm2) in laminar flow stimulated time- and amplitude-dependent increases in pinocytotic rate which returned to control levels after several hours. After 48 h continuous exposure to steady shear stress, removal to static conditions also resulted in a transient increase in pinocytotic rate, suggesting that temporal fluctuations in shear stress may influence endothelial cell function. Endothelial pinocytotic rates remained constant during exposure to rapidly oscillating shear stress at near physiological frequency (1 Hz) in laminar flow. In contrast, however, a sustained elevation of pinocytotic rate occurred when cells were subjected to fluctuations in shear stress amplitude (3-13 dyn/cm2) of longer cycle time (15 min), suggesting that changes in blood flow of slower periodicity may influence pinocytotic vesicle formation. As determined by [3H]thymidine autoradiography, neither steady nor oscillating shear stress stimulated the proliferation of confluent endothelial cells. These observations indicate that: (a) alterations in fluid shear stress can significantly influence the rate of formation of pinocytotic vesicles in vascular endothelial cells, (b) this process is force- and time-dependent and shows accommodation, (c) certain patterns of fluctuation in shear stress result in sustained elevation of pinocytotic rate, and (d) shear stresses can modulate endothelial pinocytosis independent of growth stimulation. These findings are relevant to (i) transendothelial transport and the metabolism of macromolecules in normal endothelium and (ii) the role of hemodynamic factors in the localization of atherosclerotic lesions in vivo.