Benzo[a]pyrene, which is produced during the incomplete combustion of organic material, is an abundant noxious pollutant because of its carcinogenic metabolic degradation products. The high‐affinity (K_D≈3 nm) monoclonal antibody 22F12 allows facile bioanalytical quantification of benzo[a]pyrene even in complex matrices. We report the functional and X‐ray crystallographic analysis of 22F12 in complex with 3‐hydroxybenzo[a]pyrene after cloning of the V‐genes and production as a recombinant Fab fragment. The polycyclic aromatic hydrocarbon is bound in a deep pocket between the light and heavy chains, surrounded mainly by aromatic and aliphatic amino acid side chains. Interestingly, the hapten–antibody interface is less densely packed than expected and reveals polar, H‐bond‐like interactions with the polycyclic aromatic π‐electron system, which may allow the antibody to maintain a large, predominantly hydrophobic binding site in an aqueous environment while providing sufficient complementarity to its ligand.