SH2 domain proteins transmit intracellular signals initiated by activated tyrosine kinase-linked receptors. Recent three-dimensional structures suggest mechanisms by which tandem SH2 domains might confer higher specificity than individual SH2 domains. To test this, binding studies were conducted with tandem domains from the five signaling enzymes: phosphatidylinositol 3-kinase p85, ZAP-70, Syk, SHP-2, and phospholipase C-gamma1. Bisphosphorylated TAMs (tyrosine-based activation motifs) were derived from biologically relevant sites in platelet-derived growth factor, T cell, B cell, and high affinity IgE receptors and the receptor substrates IRS-1 (insulin receptor substrate-1) and SHPS-1/SIRP. Each tandem SH2 domain binds a distinct TAM corresponding to its appropriate biological partner with highest affinity (0.5-3.0 nM). Alternative TAMs bind the tandem SH2 domains with 1,000- to >10,000-fold lower affinity than biologically relevant TAMs. This level of specificity is significantly greater than the approximately 20-50-fold typically seen for individual SH2 domains. We conclude that high biological specificity is conferred by the simultaneous interaction of two SH2 domains in a signaling enzyme with bisphosphorylated TAMs in activated receptors and substrates.