The G2A receptor (GPR132) is an orphan G-Protein Coupled Receptor (GPCR) from the “pH sensitive family” of GPCRs. It is expressed in both cells of the innate and adaptive immune system, and has a role in inflammation [1,2]. G2A is observed to be activated by oxidised fatty acids in cellular expression systems, most notably 9-Hydroxyoctadecadienoic acid (9-HODE) [3]. It is unknown if the G2A-mediated actions of 9-HODE are through direct binding to G2A, or if 9-HODE exerts an indirect action on G2A by binding to other proteins expressed within these cellular systems. In addition, effects of G2A activation on intracellular calcium signalling have only been characterised using a CHO cell expression system. Thus it is important to establish whether this is a consistent downstream signaling pathway for G2A in other cell types. Initially G2A activation was studied using a yeast assay. Unlike mammalian cell lines, yeast can be modified to express only G2A and no other receptor at the cell surface. Activation of G2A is coupled to yeast growth to give a readout for direct G2A activation. The DiscoverX PathHunter assay system gives a direct readout for receptor activation using enzyme complementation upon association of beta-arrestin 2 with G2A. Effects on intracellular calcium signalling were evaluated in rat basophilic leukemia (RBL) cells stably expressing G2A and in control RBL cells using single cell, fura- 2 microfluorimetry. We show that 9-HODE over the concentration range 1-10 uM activates the human and rat orthologues of the G2A receptor using the yeast (n=4) and beta arrestin (n=4) assays. In addition, 9-HODE (10 uM) activates calcium transients in G2A-RBL cells (n=30 cells from 3 experiments), but not control RBL cells (n=30 cells from 3 experiments). These data suggest that 9-HODE binds directly to the G2A receptor, where it can influence intracellular calcium signalling, which might contribute to its functional activity in the immune system.