A critical event in T cell receptor (TCR)-mediated signaling is the recruitment of hematopoietic-specific adaptor proteins that collect and transmit signals downstream of the TCR. Gads, a member of the Grb2 family of SH2 and SH3 domain-containing adaptors, mediates the formation of a complex between LAT and SLP-76 that is essential for signal propagation from the TCR. Here we examine the binding specificity of the Gads and Grb2 SH3 domains using peptide arrays and find that a nonproline-based R-X-X-K motif found in SLP-76 binds to the Gads carboxy-terminal SH3 domain with high affinity (K(D) = 240 +/- 45 nM). The Grb2 C-terminal SH3 domain also binds this motif, but with a 40-fold lower affinity than Gads. Single point mutations in either the relevant R (237) or K (240) completely abrogated SLP-76 association with Gads in vivo and impaired SLP-76 function. A chimeric Grb2 protein, possessing the C-terminal SH3 domain of Gads, was able to partially substitute for Gads in signaling downstream of the T cell receptor. These results provide a molecular explanation for the specific role of Gads in T cell receptor signaling, and identify a discrete subclass of SH3 domains whose binding is dependent on a core R-X-X-K motif.