Sixteen-amino-acid-long peptides, corresponding to the optimal ligand preferences of the Src homology 3 (SH3) domains of Abl, Cortactin, Crk, p53BP2, and Src, were fused to the N-terminus of Escherichia coli alkaline phosphatase (AP). These secreted fusion proteins have been used as one-step detection probes of peptide ligand-SH3 domain interactions on microtiter plates and membranes. The binding of both the class I and II SH3 ligand-AP fusion proteins to their targets is robust and specific in comparison to chemically synthesized biotinylated peptides, used either in monovalent or tetravalent formats. p53BP2 and Cortactin SH3 ligand-AP fusions have been used to screen a mouse embryo lambda cDNA expression library and resulted in the cloning of p53BP2 and several known proteins with SH3 domains similar to that of Cortactin, respectively. In addition, the approximately 60-amino-acid-long SH3 domains of Src and Abl were fused to AP and the resulting fusion proteins were found to bind specifically to their respective peptide ligands in microtiter plates and proteins containing proline-rich regions in screens of a lambda cDNA expression library. Thus, SH3 peptide ligand- and SH3 domain-AP fusion proteins are convenient and sensitive reagents for examining the specificity of SH3 domain-ligand interactions, identifying potentially interacting proteins, and establishing high-throughput screens of combinatorial chemical libraries.