Identification of the receptor binding site for ligands is an important component of studying peptide-mediated G Protein-Coupled Receptors (GPCRs). We used periodate-activated dihydroxyphenylalanine (DOPA)-modified peptides to identify specific ligand interacting residues and probe allosteric binding sites in Ste2p, a model GPCR expressed in the yeast Saccharomyces cerevisiae that binds α-factor the tridecapeptide (WHWLQLKPGQPMY) mating pheromone. Alpha-factor with DOPA substituted at residue 1 or 13 and a biotin tag on lysine at position 7 synthesized by a combination of solid-phase chain assembly and solution phase side-chain modification was used for cross-linking into Ste2p. The Bio-DOPA-α-factors cross-linked into Ste2p as demonstrated by Western blot analysis probed with neutravidin-HRP. The epitope-tagged-Ste2p-ligand complex was purified using metal ion affinity chromatography, and after cyanogen bromide treatment, avidin affinity purification was used to capture the Bio-DOPA-α-factor-Ste2p cross-linked peptide. Mass spectrometry showed crosslinking between position 13 of α-factor and residue Cys59 of Ste2p and between position 1 of α-factor and Lys269 of Ste2p. Alpha-factor truncated at the C-terminus (WHWLQLKPGQP) acted as a positive allosteric modulator of Ste2p-mediated biological activity. A biologically-active analog of the 11-mer peptide containing biotin and DOPA ([Bio-DOPA]11-mer) was synthesized and cross-linked to Ste2p resulting in a labeled product at the expected molecular weight of the receptor-pheromone complex. These studies established specific residue-to-residue interactions between Ste2p and its orthosteric ligand, revealed allosteric interactions, indicated that DOPA is an excellent probe to determine peptide hormone-receptor interactions, and demonstrated again the value of the yeast Ste2p model system for studying GPCR structure and function.