Several orphan G-protein coupled receptors, including GPR40, have recently been shown to be responsive to fatty acids. Although previous reports have suggested GPR40 detects medium and long chain fatty acids, it has been reported to be unresponsive to short chain fatty acids. In this study, we have heterologously expressed mouse GPR40 in Xenopus laevis oocytes and measured fatty acid-induced increases in intracellular calcium, via two electrode voltage clamp recordings of the endogenous calcium-activated chloride conductance. Exposure to 500µM linoleic acid (C18:2), a long chain fatty acid, stimulated significant currents in mGPR40-injected oocytes (P<0.01, ANOVA), but not in water-injected control oocytes (ns, ANOVA). These currents were confirmed as calcium-activated chloride conductances since they were biphasic, sensitive to changes in external pH, and inhibited by DIDS. Similar currents were observed with medium chain fatty acids, such as lauric acid (C12:0) (P<0.01, ANOVA), and more importantly, with short chain fatty acids, such as butyric acid (C4:0) (P<0.01, ANOVA). In contrast, no responses were observed in mGPR40-injected oocytes exposed to either acetic acid (C2:0) or propionic acid (C3:0). Therefore GPR40 has the capacity to respond to fatty acids with chain lengths of four or greater. This finding has important implications for understanding the structure:function relationship of fatty acid sensors, and potentially for short chain fatty acid sensing in the gastrointestinal tract.