We have previously reported that exposure of endothelial cells to cyclic strain elicited a rapid but transient generation of inositol 1,4,5-trisphosphate (IP₃), which reached a peak 10 s after the initiation of cyclic deformation. To address the effect of cyclic strain on intracellular Ca²⁺ concentration ([Ca²⁺]_i) and its temporal relationship to IP₃ generation, confluent bovine aortic endothelial cells were grown on flexible membranes, loaded with aequorin and the membranes placed in a custom-designed flow-through chamber. The chamber was housed inside a photomultiplier tube, and vacuum was utilized to deform the membranes. Our results indicate that the initiation of 10% average strain induced a rapid increase in [Ca²⁺]_i which contained two distinct components: a large initial peak 12 s after the initiation of stretch which closely followed the IP₃ peak, and a subsequent lower but sustained phase. Pretreatment with 5 μMGdCl₃ for 10 min or nominally Ca²⁺-free medium (CFM) for 3 min reduced the magnitude of the initial rise and abolished the sustained phase. Repetitive 10% average strain at a frequency of 60 cycles/min also elicited a single IP₃ peak at 10 s. However, there was also a large initial [Ca²⁺]_i peak followed by multiple smaller transient [Ca²⁺]_i elevations. Preincubation with 5 μMGdCl₃ or CFM diminished the initial [Ca²⁺]_i transient and markedly inhibited the late-phase component. Preincubation with 25 μM 2,5-di-(t-butyl)-1,4-benzohydroquinone (BHQ) attenuated the initial [Ca²⁺]_i transient. Cyclic-strain-mediated IP₃ formation in confluent endothelial cells at 10 s, however, was not modified by pretreatment with 25 μM BHQ, 500 μM NiCl₂, 10 nM charybdotoxin, 5 μM GdCl₃ or CFM. We conclude that in endothelial cells exposed to cyclic strain, Ca²⁺ enters the cytosol from intracellular and extracellular pools but IP₃ formation is not dependent on Ca²⁺ entry via the plasma membrane.