The production of sweat fluid by eccrine sweat glands in humans, in response to heat or exercise, is crucial to the regulation of body temperature. A key event in this process is a biphasic increase in the level of intracellular Ca2+ ([Ca2+]i) consisting of an initial agonist-induced release of Ca2+ from internal stores, followed by an influx of extracellular Ca2+ 1. We have shown previously in a cell line derived from human eccrine gland secretory cells (NCL-SG3) that the regulation of Ca2+ entry is initiated by the emptying of intracellular Ca2+ stores, and maintained through a process known as store-operated Ca2+ entry (SOCE) via the interaction of STIM and Orai proteins2. However, it is unclear if the role of STIM-Orai in these cells truly depicts the events in human eccrine secretory coils. The aim of this study was to investigate SOCE in isolated eccrine sweat gland secretory coils. Sweat glands were freshly isolated from skin samples3 obtained from individuals with consent and ethics approval. The isolated glands underwent PCR, Western blot analysis and immunofluorescence in order to identify the presence and localisation of STIM and Orai proteins. The functionality of the SOCE pathway in the isolated glands was investigated using calcium-imaging techniques in conjunction with the ER Ca2+-ATPase inhibitor thapsigargin (Tg), and a variety of known SOCE inhibitors. SiRNA knockdown of STIM and Orai proteins was used to determine relative contributions to the SOCE process. Expression for STIM1 and Orai’s 1 and 3 were confirmed by PCR and protein expression. Immunofluorescence studies showed that STIM1 was localized intracellularly (probably the ER), and Orai1 and 3 in the plasma membrane of the secretory coil cells. Fluorescence imaging experiments using the dye Fura-2, showed that the influx of extracellular Ca2+, initiated by Tg-induced emptying of the ER stores, was reduced in a dose-dependent manner with an IC50 between 10-6 and 10-7M by either diethylstilbestrol, carboxyamidotriazole or BTP2, whilst the general inhibitor 2-APB was less efficacious (IC50 approx. 10-4M). Calcium imaging of eccrine glands showed that Tg-induced SOCE was reduced ~50% in an Orai1 knockdown, whilst Orai3 knockdown had no effect on SOCE. This suggests that Orai3 can partially substitute for Orai1, and Orai1 can totally substitute for Orai3 during respective knockdowns. A double Orai1-Orai3 knockdown completely inhibited SOCE. This study demonstrates that STIM1 and Orai proteins are key components of the SOCE pathway in human eccrine sweat gland secretory cells, and are central to the activation of the membrane transport proteins that drive transepithelial chloride flux, and ultimately the production of sweat.