An analysis of the amino acid distribution at protein binding sites was carried out using 50 diverse macromolecules for which crystallographic data with a bound ligand are available. The purpose of this study is to determine whether differential trends in amino acid distributions exist at binding sites compared to other regions in the proteins. The results indicate that some residues, particularly Arg, His, Trp and Tyr are substantially more frequent at the binding sites, compared to the number of times these residues are present in proteins generally. These effects go beyond the differences seen comparing surface exposed residues to bulk protein. The resemblance in the residue utilization at the binding sites of unrelated proteins restricts the possible types of interactions with ligands, possibly accounting for the repetition of substructural motifs in chemicals with diverse pharmacological action. Further, the use of these diagnostic features may permit identification of ligand binding pockets in a protein structure deduced from sequence information or from data in the absence of a ligand. Some of these findings complement and extend previously described trends for antibody binding sites.