Thrombin and related protease-activated receptors1, 2, 3 and 4 (PAR1-4) play a multifunctional role in many types of cells including endothelial cells. Here, using RT-PCR and immunofluorescence staining we showed for the first time that PARs1-4 are expressed on primary human brain microvascular endothelial cells (HBMEC). Digital fluorescence microscopy and fura-2 were used to monitor intracellular Ca²⁺ changes ([Ca²⁺]_i) in response to thrombin and PAR1-activating peptide (PAR1-AP), SFFLRN. Both thrombin and PAR1-AP induced a dose-dependent [Ca²⁺]_i rise that was inhibited by pretreatment of HBMEC with phospholipase C inhibitor, U73122, and (sarco)endoplasmic Ca²⁺-ATPase inhibitor, thapsigargin. Thrombin induced transient [Ca²⁺]_i increase, while PAR1-AP exhibited sustained rise of [Ca²⁺]_i. The PAR1-AP-induced sustained [Ca²⁺]_i rise was significantly reduced in the absence of extracellular calcium or in the presence of an inhibitor of store-operated calcium channels, SKF96365. Restoration of extracellular calcium to the cells that were initially activated by PAR1-AP in the absence of extracellular Ca²⁺ resulted in significant [Ca²⁺]_i rise, however, this effect was not observed after thrombin stimulation. Pretreatment of the cells with a low concentration of thrombin (0.1 nM) prevented [Ca²⁺]_i rise in response to high concentration of thrombin (10 nM), but pretreatment with PAR1-AP did not prevent subsequent [Ca²⁺]_i rise to high concentration of PAR1-AP. Additionally, treatment with thrombin decreased transendothelial electrical resistance in HBMEC while PAR1-AP was without significant effect. These findings suggest that, in contrast to thrombin, stimulation of PAR1 by untethered peptide SFFLRN results in stimulation of store-operated calcium influx without significantly affecting the brain endothelial barrier functions.