Human bestrophin 1 (hBest1), which is genetically linked to several kinds of retinopathy and macular degeneration in humans, was proposed to be a Ca²⁺ activated Cl^- channel and a regulator of intracellular Ca²⁺ signaling. We demonstrate that hbest1 is localized in the endoplasmic reticulum (ER) where it interacts with stromal interacting molecule 1 (Stim1), the ER-Ca²⁺ sensor that controls store operated Ca²⁺ entry (SOCE). Intracellular Ca²⁺ transients induced by stimulation of purinergic P2Y2-receptors in HEK293 cells were more transient after expression of hbest1, but were less transient after overexpression of dominant negative hbest1-R218C. By means of immunoprecipitation 2D gel- and MALDI TOF –analysis, we identified p21 activated kinase 2 (Pak2) as an interaction partner for hBest1. Pak2 was found to phosphorylate hBest1, thereby enhancing Ca²⁺ signaling and activation of Ca²⁺ dependet Cl^- (TMEM16A) and K⁺ (SK4) channels. Lack of bestrophin1 expression in respiratory epithelial cells of mBest1 knockout mice caused expansion of ER cisterns that showed Ca²⁺-oxalate deposits. We propose that hbest1 is important for Ca²⁺ handling of the ER store, probably by controlling the function of Stim1 and/or by acting as a counter-ion channel to balance transient potentials occurring through refill of the ER-Ca²⁺ store or inositol triphosphate (IP₃) induced Ca²⁺ release. Thus bestrophin 1 controls activation of Ca²⁺ dependet ion channels by regulation of compartmentalized Ca²⁺ signaling. Supported by DFG SFB699 A6/A7