Engagement of the TCR by specific antigen results in activation of a tyrosine kinase pathway. A candidate for the kinase responsible for the rapid tyrosine phosphorylation detected with T cell activation Is p60fyn, a member of the src kinase family. In an earlier study [Samelson et a/. (1990) Proc. Natl Acad. ScL USA 87: 4358] this enzyme was co-lmmunoprecipltated with the TCR from T cells solubillzed In digitonin. In that study a sensitive in vitro kinase assay was used to detect the associated p60fyn. It was subsequently found that the reproduclblllty of the interaction depended on lot-to-lot variations in digitonin. To eliminate the possibility that the association of antigen receptor and kinase is an artifact of solubilizatlon with Ill-defined digitonin preparations, a cross-linking protocol was developed to stabilize the interaction between the TCR and p60fyn. T cells were permeablllzed with tetanolysln and proteins were cross-linked with the water soluble chemical cross-linker, 3, 3' dlthiobis(sulfosucclnlmldylpropionate). These experiments allowed the confirmation of the interaction between the TCR, p60fyn and several additional proteins. The cross-linking studies also enabled the mapping of the Interaction of p60fyn and associated proteins to the TCR ζ-chaln. This technique should have a general use In stabilizing interactions between other receptors and molecules required for intracellular signaling.