Meltrin alpha/ADAM12 is a member of the ADAM/MDC family proteins characterized by the presence of metalloprotease and disintegrin domains. This protein also contains a single transmembrane domain and a relatively long cytoplasmic domain containing several proline-rich sequences. These sequences are compatible with the consensus sequences for binding the Src homology 3 (SH3) domains. To determine whether the proline-rich sequences interact with SH3 domains in several proteins, binding of recombinant SH3 domains to the meltrin alpha cytoplasmic domain was analysed by pull-down assays. The SH3 domains of Src and Yes bound strongly, but that of Abl or phosphatidylinositol 3-kinase p85 subunit did not. Full-length Grb2/Ash bound strongly, whereas its N-terminal SH3 domain alone did less strongly. Src and Grb2 in bovine brain extracts also bound to meltrin alpha cytoplasmic domain on affinity resin. Furthermore, immunoprecipitation with a monoclonal antibody to meltrin alpha resulted in coprecipitation of Src and Grb2 with meltrin alpha in cell extracts, suggesting that Src and Grb2 are associated in vivo with meltrin alpha cytoplasmic domain. This notion was also supported by the findings that exogenously expressed meltrin cytoplasmic domain coexisted with Src and Grb2 on the membrane ruffles. The C-terminal Tyr901 of meltrin alpha was phosphorylated both in vitro and in cultured cells by v-Src. These results may imply that meltrin alpha cytoplasmic domain is involved in a signal transduction for some biological function through the interaction with SH3-containing proteins.