Brain capillary endothelial cells (BCECs) form the blood brain barrier (BBB), which is essential for maintaining homeostasis of the brain. Net cellular turnover, which results from the balance between cell death and proliferation is important in maintaining BBB homeostasis. Here we report a novel mechanism which underlies ATP-induced cell proliferation in t-BBEC 117, a cell line derived from bovine brain endothelial cells. Application of 0.1 - 30 µM ATP to t-BBEC 117 concentration-dependently increased intracellular Ca2+ concentration ([Ca2+]i) in two phases; an initial transient phase and a later and smaller sustained one. These two phases of [Ca2+]i rise were mainly due to Ca2+ release and sustained Ca2+ influx, respectively. The pretreatment with apamin, a selective blocker of small conductance Ca2+ activated K+ channels (SK), significantly reduced both the [Ca2+]i increase and K+ current induced by ATP. Transcripts of corresponding to P2Yx, SK2 and transient receptor potential channels (TRP) were detected in t-BBEC 117. Knock-down of SK2 protein, which was the predominant Ca2+ activated K+ channel expressed in t-BBEC 117, by siRNA significantly reduced both the sustained phase of the [Ca2+]i rise and the K+ current induced by ATP. Cell proliferation was increased significantly by the presence of the stable ATP analogue (ATPS). This effect was blunted by UCL1684, a synthesized SK blocker. In conclusion, in brain endothelial cells ATP-induced [Ca2+]i rise activates SK2 current, and the subsequent membrane hyperpolarization enhances Ca2+ entry presumably through TRP channels. This positive feedback mechanism can account for the augmented cell proliferation by ATP.