Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) interact with VPAC₂ receptors in rabbit and guinea pig (GP) gastric muscle but with functionally distinct VIP and PACAP receptors in GP tenia coli. This study examined whether selectivity for VIP was determined by two residues (40, 41) in the extracellular domain that differ in the VIP receptors of GP gastric and tenial muscle. A mutant rat VPAC₂ receptor (L40F, L41F), and two chimeric receptors in which the NH₂-terminal domain of rat VPAC₂ receptor was replaced with that of GP gastric (chimeric-G) or tenia coli (chimeric-T) VIP receptors, were constructed and expressed in COS-1 cells. VIP and PACAP bound with equal affinity to wild-type and mutant rat VPAC₂ receptors and to chimeric-G receptor (IC₅₀: VIP 0.3 ± 0.1 to 1.5 ± 0.4 nM, PACAP 0.4 ± 0.1 to 2.5 ± 0.1 nM) and stimulated cAMP with equal potency (EC₅₀: VIP 13 ± 5 to 48 ± 8 nM, PACAP 8 ± 3 to 31 ± 14 nM). VIP bound with high affinity also to chimeric-T receptor (IC₅₀: 0.5 ± 0.1 nM) and stimulated cAMP with high potency (EC₅₀: 3 ± 1 nM). In contrast, PACAP exhibited >1,000-fold less affinity for binding or potency for stimulating cAMP. We conclude that GP tenia coli express a VIP-specific receptor and that selectivity is determined by a pair of extracellular phenylalanine residues.