Human CD7 is a 40 kDa protein expressed on thymocytes, early T, B, NK and myeloid lineage cells in bone marrow, and on mature T and NK cells. Previous studies suggested human CD7 may be involved in T and NK cell activation and/or adhesion, and that CD7-mediated cell activation may be transduced via the lipid kinase phosphatidylinositol 3-kinase (Pi3-kinase), a heterodimeric cytosolic protein consisting of an 85 kDa adaptor subunit that is coupled to a 110 kDa catalytic subunit. It has recently been shown that a sequence motif present in the cytoplasmic tall of both human and mouse CD7 bound with high affinity to recombinant SH2 domains present in the p85 subunit of Pi3-kinase. In this work, we used co-precipitation with anti-CD7 mAb 3A1 and recombinant p85 SH2-GST fusion proteins and peptide competition analysis to demonstrate that the cytoplasmic tail of CD7 interacts with a functional Pi3-kinase via the pTyr-X-X-Met motif. Furthermore, we show that cross-linking of CD7 markedly increased the amount of Pi3-kinase activity associated with CD7. The interaction of CD7 with the Pi3-kinase signal transduction pathway provides a mechanism for the previously observed functional responses attributed to CD7-mediated T and NK cell activation.