In the innate immune defense, professional phagocytes such as neutrophils, monocytes and macrophages play an important role in clearance of pathogenic intruders and in triggering the adaptive immune system. Monocytes invade tissues as a result of chemokine attraction wherein they differentiate into macrophages and dendritic cells and act as first line of host defense. Store-operated calcium entry (SOCE) through calcium release activated calcium (CRAC) channels composed of Orai1-3 and STIM1 and 2 proteins is implicated in a plethora of cellular functions. While a major role for CRAC channels was revealed especially in T cells and neutrophils, it is presently not known if and what role Orai and STIM proteins play in human monocytes. For efficient immune response, monocytes need to generate high amounts of oxidants upon encounter with an antigen. Hence, we investigated the role of Orai and STIM isoforms on reactive oxygen species (ROS) production, Ca2+ homeostasis, and the effects of their interplay for monocyte function. Expression analysis using qRT-PCR showed the presence of all CRAC channel components in human monocytes, with comparatively high Orai3 and STIM2 levels. To determine the interplay of SOCE and ROS production, monocytes were activated by internal Ca2+ store depletion using thapsigargin or bacterial products (fMLF). To determine the specific contribution of a particular CRAC channel component in monocyte SOCE we used small interfering RNA gene silencing. Our measurements showed a significant contribution of Orai1 and STIM1 and, in contrast to other phagocytes, also Orai3 and STIM2 proteins. Not unexpectedly, NADPH-oxidase 2 (NOX2) was identified as the main enzymatic source of ROS in monocytes, which showed a clear dependency on not only internal Ca2+ concentration, but also on SOCE. To determine the dependency of NOX2 on Ca2+ and thus Orai and STIM, we analyzed quantitative and temporal parameters of ROS generation using fluorescent dyes and electronic paramagnetic resonance (EPR) spectroscopy. Down-regulation of Orai1, STIM1 and STIM2 expression levels resulted in altered kinetics and a decrease in ROS production. Contrary to these results, lower levels of Orai3 led to enhanced SOCE and consequently increased ROS production. We demonstrate the existence of a regulatory feedback loop between Orai channels and NOX2 enzymes essential for monocyte function. Based on our preliminary data we are currently investigating the contribution of Orai and STIM proteins in monocyte migration and invasion and phagocytosis.