1. Several combinations of cysteine to serine mutations at positions 57, 93, 99 and 129 in the extracellular N-terminal domain of human metabotropic 5a (hmGlu5a) receptors were produced and expressed in HEK293 cells. Quisqualic acid-induced intracellular calcium ([Ca(2+)](i)) mobilization and inositol phosphates (IP) accumulation revealed an apparent increased efficacy and decreased potency for hmGlu5a mutants C57S, C99S and C57/99S, as well as a total loss of function for the mutant C57/93/99/129S. 2. [(3)H]Quisqualate saturation analysis revealed mutants C57S, C99S, C57/99S and the tetramutant C57/93/99/129S to have unchanged K(D) but reduced B(max) values. [(3)H]MPEP saturation analysis on the same membrane preparations revealed no difference in K(D) for any mutant, but a decrease in B(max) value for the C57/93/99/129S receptor. 3. Inverse agonism of MPEP at hmGlu5a receptors was partially reduced by mutation C57S, significantly reduced by C99S and C57/99S mutations and totally abolished in the tetramutant. 4. We confirmed the surface expression of all the mutated receptors using [(3)H]MPEP binding analysis on whole cells. However, B(max) values were increased for mutant C57S, C99S, and C57/99S but decreased in the C57/93/99/129S receptor. 5. The 24 h preincubation of cells expressing hmGlu5a receptors with 1 muM MPEP followed by extensive washing dramatically increased the wild-type receptor efficacy to quisqualate, to the same levels seen with C57/99S receptors. MPEP preincubation did not affect C57/99S function. 6. We conclude that cysteines 57 and 99 are key residues necessary for modulating hmGlu5a receptor function.