Ca2+ signaling depends on a tight regulation of the intracellular Ca2+ concentration. Alterations in basal Ca2+ can lead to various diseases and likely contribute to development of abnormal growth. Different regulators such as calmodulin and Ca2+ pumps limit cytosolic [Ca2+] and their down-regulation by siRNA lead to an increased basal [Ca2+]. Another important regulator is the stromal interaction molecule 2 (STIM2) that shows a reduction in basal [Ca2+] following knock down. The two known isoforms of STIM, STIM1 and STIM2, are ER resident membrane proteins which sense the Ca2+ content of the ER via their luminal EF-hands. After partial or complete store depletion STIM-proteins multimerize and trigger store-operated calcium entry (SOCE) by directly gating Orai channels localized at the plasma membrane. Here, we report the identification and characterization of a novel STIM2 splice variant, named STIM2.1, which differs in a single additional exon consisting of only 8 amino acids located within the STIM2 channel activating domain (CAD). We show the novel variant STIM2.1 is present in a variety of primary cells and cell lines although its relative expression varies in regard to the known variant (STIM2.2) and depends on the activation state and cell type. In contrast to STIM2.2, STIM2.1 is unable to gate Orai channels. Coexpression of STIM2.1 together with STIM1 displays reduced SOCE when compared to STIM2.2 coexpression. Splice variant specific knockdown of STIM2.1 in naive human CD4+ T cells increases SOCE whereas specific down-regulation of STIM2.2 decreases basal calcium as well as SOCE, suggesting that STIM2.1 acts as a negative regulator of STIM mediated SOCE. Biochemical experiments are being conducted to delineate the functional defects of STIM2.1.