Mechanisms controlling endoplasmic reticulum (ER) Ca2+ homeostasis are important regulators of resting cytoplasmic Ca2+ concentration ([Ca2+](cyto)) and receptor-mediated Ca2+ signalling. Here we investigate channels responsible for ER Ca2+ leak in THP-1 macrophage and human primary macrophage. In the absence of extracellular Ca2+ we employ ionomycin action at the plasma membrane to stimulate ER Ca2+ leak. Under these conditions ionomycin elevates [Ca2+]cyto revealing a Ca2+ leak response which is abolished by thapsigargin. IP3 receptors (Xestospongin C, 2-APB), ryanodine receptors (dantrolene), and translocon (anisomycin) inhibition facilitated ER Ca2+ leak in model macrophage, with translocon inhibition also reducing resting [Ca2+](cyto). In primary macrophage, translocon inhibition blocks Ca2+ leak but does not influence resting [Ca2+](cyto). We identify a role for translocon-mediated ER Ca2+ leak in receptor-mediated Ca2+ signalling in both model and primary human macrophage, whereby the Ca2+ response to ADP (P2Y receptor agonist) is augmented following anisomycin treatment. In conclusion, we demonstrate a role of ER Ca2+ leak via the translocon in controlling resting cytoplasmic Ca2+ in model macrophage and receptor-mediated Ca2+ signalling in model macrophage and primary macrophage. (C) 2017 Elsevier Inc. All rights reserved.