Immunoglobulin receptor family tyrosine-based activation motifs (ITAM) define a conserved signaling sequence, EX2YX2L/IX7YX2L/I, that mediates coupling of the T cell antigen receptor (TCR) to protein tyrosine kinases (PTK). In the present study, we explored the role of phosphorylation of the two ITAM tyrosine residues in the interactions of the motif with the PTK ZAP-70 and p59fyn. The data show that the phosphorylation of a single tyrosine within the motif enables binding of p59fyn, whereas phosphorylation of both tyrosines within the motif is required for maximal binding of the PTK ZAP-70. Quantitative binding experiments show that nanomolar concentrations of the doubly phosphorylated zeta 1-ITAM are sufficient for ZAP-70 recruitment, whereas micromolar levels of singly phosphorylated ITAM are necessary for p59fyn binding. ZAP-70 binds with low efficiency to a singly phosphorylated ITAM, but shows preferential binding to the C-terminal phosphotyrosine in the ITAM, whereas p59fyn binds selectively to the N-terminal phosphotyrosine. The present data thus show that there is the potential for a singly phosphorylated ITAM to couple to cellular PTK. Moreover, the data suggest a mechanism for heterogeneity in signal transduction responses by the TCR, since ITAM could differentially couple the TCR to downstream signaling events depending on their phosphorylation state.