To test the hypothesis that the determinants for agonist selectivity of class III metabotropic glutamate receptors (mGluRs) are localized in the N-terminal extracellular domain, a chimaeric cDNA was constructed where 519 amino acids of the N-terminal extracellular domain of human mGluR1b were exchanged with the corresponding region of human mGluR4. The pharmacological profile of the chimaera, designated hmGlu(R4)1-519/1b, was analysed by recordings of intracellular calcium concentration ([Ca2+]i) in transiently transfected HEK 293 cells and compared with that of human mGluR1b and human mGluR4a stably expressed in Chinese hamster ovary cells. Application of 100 microM L-2-amino-4-phosphonobutyrate (L-AP4), a class III mGluR-specific agonist, induced a rise in [Ca2+]i in hmGlu(R4)1-519/1b but not in hmGluR1b expressing cells. In contrast, application of quisqualate (100 microM) induced a rise in [Ca2+]i at hmGluR1b but not at hmGlu(R4)1-519/1b. Dose-response analysis with L-AP4 and L-glutamate at hmGlu(R4)1-519/1b revealed a half-maximal effect (EC50) of 16.0 microM and 196 microM, respectively. The EC50 values for quisqualate, glutamate and (1S,3R)-ACPD at hmGluR1b were 10.25 microM, 225 microM and 3060 microM, respectively. The rank order of agonist potency of hmGlu(R4)1-519/1b corresponds to that of hmGluR4 (L-AP4 > L-glutamate > (1S,3R)-ACPD > quisqualate) but is different from that of hmGluR1b (quisqualate > glutamate >> (1S,3R)-ACPD).