Immunoglobulin family tyrosine-based activation motifs (ITAM), which define the conserved signaling sequence EX2YX2L/IX7YX2L/I, couple the T cell antigen receptor (TCR) to cellular proteins including protein tyrosine kinases (PTK) and adapter molecules. The TCR is a multichain complex with four invariant chains CD3 gamma, delta and epsilon that each contain a single ITAM and the TCR zeta chain that contains three ITAM. The present study explores the protein interactions of the doubly phosphorylated CD3 gamma, delta, epsilon ITAM to determine whether they have common or unique biochemical properties. The data show that the doubly phosphorylated ITAM all bind the PTK ZAP-70, but the ITAM also variably bind the PTK p59fyn and the adapters Shc, Grb-2 and the p85 regulatory subunit of phosphoinositol 3' kinase. The CD3 and zeta ITAM display a hierarchy of ZAP-70 binding: zeta 1 = gamma = delta > zeta 3 > zeta 2 = epsilon. Shc, Grb-2 and p85 could bind the zeta ITAM and the CD3 gamma and delta ITAM, but not the CD3 epsilon ITAM. There were also subtle differences in the hierarchy of reactivity of these adapters for the CD3 gamma, delta and zeta ITAM that show that the zeta, CD3 gamma, delta and epsilon ITAM have different binding properties. The present study thus shows that the different ITAM of the TCR/CD3 complex can interact with different cytosolic effectors, indicating that differential ITAM phosphorylation during T cell activation could be a mechanism to generate signaling diversity by the TCR complex.