The Src-homology-3 domain (SH3) is an evolutionarily conserved, 50- to 60-amino-acid module carried by intracellular proteins involved in the transduction of signals for cell polarization, motility, enzymatic activation, and transcriptional regulation. The SH3 drives protein-protein interactions through binding to proline-rich ligands. This function relies on the conserved secondary structure, whereas the SH3 primary structure is highly diverse. Taking advantage of the fact that the few conserved amino acids are clustered near the N- and C-terminal ends, we designed degenerate oligonucleotides spanning these two regions and screened by PCR a variety of normal and tumor tissues for the expression of SH3-containing transcripts. Using this strategy, we have identified a novel SH3-containing human gene family of six related transcripts that map to four different chromosomes. The SH3 domain lies at the C-terminal end and shows 56-50% amino acid homology to the C-terminal SH3 of Sem-5/Drk/GRB2. The N-terminal segment of this novel SH3GL (from SH3-containing Grb2-like) gene family does not resemble any known protein. Three of these transcripts are in-frame and show a peculiar tissue distribution: SH3GL2 is preferentially expressed in the brain, SH3GL3 in brain and testis, and SH3GL1 is ubiquitous.