HIV gp120 binding to CD4 suppresses TCR function. The molecular mechanism of this anergizing effect is incompletely understood. Studies reported here reveal that CD4 ligation initiates p56(lck) activation and renders human peripheral T cells and HPB-ALL cells hyporesponsive to Ag receptor stimulation, as indicated by the failure of TCR binding ligands to induce either protein tyrosine phosphorylation or elevation of intracellular-free calcium concentration. To approach the possibility that p56(lck)-mediated tyrosine phosphorylation of specific sites within TCR ζ-chain might be involved in the gp120-induced TCR signaling defect, we tested the kinase's ability to phosphorylate various ζ peptides. Kinetic analyses indicate that peptides derived from the in vitro autophosphorylation site of p56(lck) Y394 and two sites within ζ, Y84, and Y152, are equally effective substrates for p56(lck), whereas p59(lyn) prefers a substrate peptide derived from a different site within ζ Y142. Although these data are consistent with the possibility that gp120-mediated signal disruption of TCR could be due to p56(lck) phosphorylation of Y84 and Y152 residues within ζ, further experiments revealed that gp120 does not induce detectable ζ tyrosine phosphorylation under conditions in which it disrupts TCR signaling. These data indicate that gp120 can induce uncoupling of TCR from the earliest events in signal transduction and that this effect can be mediated by a mechanism other than tyrosine phosphorylation of TCR ζ-chain.