Split-pool synthesis was used to prepare large numbers of spatially-separated molecules and thereby to investigate the specificity pockets of similar SH3 domains found in the tyrosine kinases Src and lick. By taking into account the structure of the Src SH3 domain complexed to a ligand containing non-peptide-binding elements, the molecules were designed to complement the topography of the protein's binding pocket. This procedure led to the discovery of ligands having greater affinity and enhanced selectivity for the Src SH3 domain. It also yielded non-natural ligands that bind selectively to the Hck SH3 domain. Insights gained from this strategy may facilitate the discovery of molecules useful for evaluating the cellular function of SH3 domain-containing proteins.