VIP receptors belong to a subfamily of G protein-coupled receptors that includes secretin, glucagon, PTH and several other receptors. We have used site-directed mutagenesis to investigate the requirement of some highly conserved residues in the extracellular loops including aspartate 196 (mutant D196A), leucine 199 (mutant L199A), tryptophane 286 (mutant W286A) and tryptophane 294 (mutant W294A) for the ability of the human VIP1 receptor to bind VIP and to mediate VIP-stimulated cAMP production. After transfection of mutated cDNAs in Cos-7 cells, it appeared that 1) mutants L199A, W286A and W294A bound VIP with the same dissociation constant as the wild-type receptor whereas mutant D196A did not bind 125I-VIP; 2) mutants L199A, W286A and W294A mediate VIP-stimulated cAMP production with the same EC50 as the wild-type receptor whereas VIP displayed a 500-fold decrease of potency in promoting cAMP production through mutant D196A. Since all mutated receptor proteins were expressed and delivered at the plasma membrane (immunofluorescence studies), it is concluded that the first extracellular loop of the human VIP1 receptor contains a highly conserved aspartate residue which is essential for VIP binding and VIP-stimulated cAMP production.