The neurotransmitter GABA (gamma-aminobutyric acid) functions as the major inhibitory neurotransmitter in the central nervous system of vertebrates and invertebrates. In vertebrates GABA signals both through ionotropic receptors (GABA(A), GABA(C)), which induce fast synaptic inhibitory responses, and through metabotropic receptors (GABA(B)), which play a fundamental role in the reduction of presynaptic transmitter release and postsynaptic inhibitory potentials. Whilst GABA(A) and GABA(C) receptors have been cloned from vertebrates as well as invertebrates, GABA(B) receptors have only been identified in vertebrate species to date, although indirect evidence suggests their existence in arthropods, too. Here we report the cloning of three putative invertebrate GABA(B) receptor subtypes (D-GABA(B)R1, R2 and R3) isolated from Drosophila melanogaster. Whilst D-GABA(B)R1 and R2 show high sequence identity to mammalian GABA(B)R1 and R2, respectively, the receptor D-GABA(B)R3 seems to be an insect-specific subtype with no known mammalian counterpart so far. All three D-GABA(B)R subtypes are expressed in the embryonic central nervous system. In situ hybridization of Drosophila melanogaster embryos shows that two of the D-GABA(B)Rs (D-GABA(B)R1 and R2) are expressed in similar regions, suggesting a coexpression of the two receptors, whilst the third D-GABA(B)R (D-GABA(B)R3) displays a unique expression pattern. In agreement with these results we have only been able to functionally characterize D-GABA(B)R1 and R2 when the two subtypes are coexpressed either in Xenopus laevis oocytes or mammalian cell lines, whilst D-GABA(B)R3 was inactive in any combination. The pharmacology of the coexpressed D-GABA(B)R1/2 receptor was different from the mammalian GABA(B)Rs: e.g. baclofen, an agonist of mammalian GABA(B)Rs, showed no effect.