Stacking interactions in nitroarene binding sites of proteins were studied through analyses of structures in the protein data bank (PDB), as well as DFT and ab initio computations applied to model systems. Stacked dimers of mono-, di-, and trinitrobenzene with the amino acid side chains histidine (His), phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp) were optimized at the B97-D/TZV(2d,2p) level of theory. Binding energies for the global minimum dimer geometries were further refined at the estimated CCSD(T)/aug-cc-pVTZ level of theory. The results show that the interactions between aromatic amino acids and nitroarenes are very strong (up to -14.6 kcal mol(-1)), and the regiochemistry of the nitro substituents plays a significant role in the relative monomer orientations and strength of the interaction. In contrast to model stacked benzene dimers, effects of nitro substituents in stacking complexes with aromatic amino acid side chains are not perfectly additive. This is attributed to direct interactions of the nitro substituents with functional groups in the amino acid side chain. Overall, the strength of stacking interactions with these nitrobenzenes follows the order Trp > Tyr > Phe approximate to His. We also analyzed nitroarene binding sites in the PDB. Out of 216 selected crystal structures containing nitroarene ligands, 191 have nearby aromatic residues, providing 65 examples of pi-stacking interactions involving a nitroarene. Of these, the representations of the different aromatic amino acids (Trp > Tyr > Phe > His) are correlated with the strength of model complexes of nitroarenes, with the exception of His. B97-D computations applied to complexes extracted from these crystal structures reveal that pi-stacking interactions between the nitroarene and aromatic amino acid side chains exhibit a broad range of strengths, with many contributing significantly to binding.