Our objective was to identify novel ligands for members of the family of G protein-coupled receptors that responds to free fatty acids, to define their selectivity and mode of binding and to explore their use to define the specific roles of the individual receptors. A series of small carboxylic acids were assessed for their capacity to activate human FFA2 and FFA3 using transfected cell lines and the mode of binding of these further assessed following expression of mutated receptors. These were then tested for activity at each of rat, mouse and bovine orthologues of the receptors. Cells lines endogenously expressing either or both FFA2 and FFA3 were identified via RT-PCR and the selectivity of the ligands then assessed in such cells. A series of small carboxylic acids with selectivity for either human FFA2 or FFA3 were identified and shown to maintain this selectivity at a range of species orthologues as well as in cell lines endogenously expressing these receptors. These ligands will be useful tools in exploring the biology and function of these two closely related receptors in both ex vivo and in vivo models of diabetes, adiposity and inflammation.