The authors have constructed a vast library of peptides to identify compounds that bind to antibodies and other receptors. They expressed millions of different hexapeptides at the N-terminus of the adsorption protein (pIII) of fd phage using the vector fAFF1, which allows cloning of oligonucleotides in various locations within gene III. A library of 3 × 10^8 recombinants was generated by cloning randomly synthesized oligonucleotides. The library was screened for high-avidity binding to a monoclonal antibody (3-E7) specific for the N-terminal sequence of β-endorphin (Tyr-Gly-Gly-Phe). After three rounds of affinity purification, 51 clones were selected and sequenced, revealing that all contained tyrosine as the N-terminal residue and glycine as the second residue. The binding affinities of six chemically synthesized hexapeptides from this set ranged from 0.35 μM to 8.3 μM, compared to 7.1 nM for a known high-affinity ligand. This study demonstrates that ligands can be identified without prior knowledge of antibody specificity, and peptide libraries are likely useful for finding ligands that bind to other receptors and discovering pharmacological agents.The authors have constructed a vast library of peptides to identify compounds that bind to antibodies and other receptors. They expressed millions of different hexapeptides at the N-terminus of the adsorption protein (pIII) of fd phage using the vector fAFF1, which allows cloning of oligonucleotides in various locations within gene III. A library of 3 × 10^8 recombinants was generated by cloning randomly synthesized oligonucleotides. The library was screened for high-avidity binding to a monoclonal antibody (3-E7) specific for the N-terminal sequence of β-endorphin (Tyr-Gly-Gly-Phe). After three rounds of affinity purification, 51 clones were selected and sequenced, revealing that all contained tyrosine as the N-terminal residue and glycine as the second residue. The binding affinities of six chemically synthesized hexapeptides from this set ranged from 0.35 μM to 8.3 μM, compared to 7.1 nM for a known high-affinity ligand. This study demonstrates that ligands can be identified without prior knowledge of antibody specificity, and peptide libraries are likely useful for finding ligands that bind to other receptors and discovering pharmacological agents.