A common RXL motif waa found in proiine-rich iigands that were selected from a blased combinatorial peptide library on the basis of their ability to bind specifically to the SH3 domains from phosphatidyllnosltoi Skinase (Pl3K) or c-Src. The solution structure of the Pl3K SH3 domain complexed to one of these ligands, RKLPPRPSK (RLPl), was determined. Shucture-based mutations were introduced into the Pi3K SH3 domain and the RLPl ligand, and the influence of these mutations on blndlng was evaluated. We conclude that SH3 domains recognixe proline-rich motifs possesslng the left-handed type II poiyproilne (PPII) helix conformation. Two proilne residues directly contact the recep tor. Other proiines in the iigands appear to function as a molecular scaffold, promoting the formation of the PPII helix. Three nonproiine residues consisting of combinationsof arginlne and leucine interact extensively wlth the SH3 domain and appeartoconfer ilgand specificity.