The SH3 domain has been shown recently to bind peptide sequences that lack the canonical PxxP motif. The diverse specificity in ligand recognition for a group of fifteen SH3 domains have now been investigated using arrays of peptides derived from the proline-rich region of the SH2 domain-containing leukocyte protein of 76 kDa (SLP-76). Screen of the peptide arrays using individual or mixed SH3 domains has allowed the identification of a number of candidate SH3-binding peptides. While some peptides contain the conventional PxxP motif, most are devoid of such a motif and are, instead, enriched in basic residues. Fluorescent polarization measurements employing soluble peptides and purified SH3 domains demonstrated that several SH3 domains, including those from Grb2, NCK and PLC-1, bound with moderate affinities (10 to 100 M) to a group of non-conventional peptides. Of particular interest, the PLC-1 SH3 domain was found to associate with SLP-76 through at least three distinct sites, two of which bore a novel KKPP motif and the other contained the classic PxxP sequence. Intriguingly, mutation of critical residues for the three sites not only affected binding of SLP-76 to the PLC-1 SH3 domain, but also to the Grb2 SH3-C domain, indicating that the binding sites in SLP-76 for the two SH3 domains are overlapped. Our studies suggest that the SH3 domain is an inherently promiscuous interaction module capable of binding to peptides that may or may not contain a PxxP motif. Furthermore, the identification of numerous non-conventional SH3-binding peptides in SLP-76 implies that the global ligand pool for SH3 domains in a mammalian proteome may be significantly greater than previously acknowledged.