X-ray crystallographic techniques were used to study the modes of binding of N-formylated chemotactic peptides to the Mcg light chain (Bence-Jones) dimer. By difference Fourier analyses at 2.7-A resolution four N-formylated tripeptides were found to occupy similar sites in the main binding cavity of the dimer. In all cases the N-formyl group appeared to form a hydrogen bond with a phenolic hydroxyl group of a tyrosine residue (No. 38, monomer 1) at the base of the cavity. N-formylation was necessary, since di-, tri- and tetrapeptides with free alpha-amino groups failed to bind. Although methionine in ligand position 1 was optimal for binding, it could be replaced with norleucine. Position 2 was less critical, providing the side chain was bulky and hydrophobic (e.g. leucine, methionine or phenylalanine). An aromatic residue like phenylalanine was most favorable in position 3. These bound ligands were site-filling and wedge-shaped, with their side chains swept back toward the entrance of the cavity to conform to the space available for binding. In the binding site side chains and polypeptide segments of the hypervariable loops also moved in ways improving the complementarity between protein and ligand. The stereochemical requirements for binding were markedly similar to those found in interactions of neutrophil receptors with the same series of tripeptides. An N-formylated dipeptide, N-f-Met-Trp, was bound with equal occupancies in two overlapping subsites. In the deeper site the N-formyl group and methionine side chain were situated in positions comparable to those in the N-formyl tripeptides, but the peptide bond between methionine and tryptophan was in the cis configuration. In the outer site the corresponding peptide bond was in the energetically more favourable trans configuration.