The CD7 cluster of mAb identifies a 40-kDa glycopolypeptide that is present on a major subset of human T cells. CD7 Ag mediates an accessory pathway of T cell activation in that cross-linked CD7 mAb are mitogenic, and signals delivered via CD7 Ag stimulate integrin-mediated adhesion. We have found that the CD7 molecule is associated with a tyrosine kinase whose major substrate is CD45. In vitro phosphorylation of CD7, CD3, or CD45 immunoprecipitates prepared from lysates of human T cells showed a similar pattern of multiple phosphorylated polypeptides; in addition, these immunoprecipitates phosphorylated a tyrosine kinase-specific peptide. Surface-iodinated T cells were lysed and immunoprecipitated with CD7, CD3, and CD45 mAb. Bands characteristic of CD45 and CD3 were identified in CD7 immunoprecipitates. Confirmation of an association of CD7 with CD3 and CD45 was obtained from Western blotting and fluorescence resonance energy transfer experiments. Furthermore, we provide evidence using immunoprecipitation and Western blotting that CD7 exists as a homodimer. These data support the hypothesis that CD7 exists in an oligomeric complex with CD3/TCR, the protein tyrosine phosphatase CD45, and a tyrosine kinase, thereby providing a physical basis for the accessory role of the CD7 molecule in T cell activation.