The rapid and reversible upregulation of the functional activity of integrin receptors on T lymphocytes is a vital step in the adhesive interactions that occur during successful T cell recognition of foreign antigen and transendothelial migration. Although the ligation of several different cell surface receptors, including the antigen-specific CD3/T cell receptor complex, the CD2, CD7, and CD28 antigens, as well as several chemokine receptors, has been shown to rapidly upregulate integrin function, the intracellular signaling events that initiate this increase in adhesion remain poorly defined. In this study, we have used DNA-mediated gene transfer to explore the role of phosphatidylinositol 3-kinase (PI 3-K) in the upregulation of β1 integrin functional activity mediated by the CD2 antigen. CD2 was expressed in the myelomonocytic cell line HL60, which expresses β1 integrins that mediate adhesion to fibronectin and VCAM-1 in an activation- dependent manner. Antibody stimulation of CD2 expressed on HL60 transfectants resulted within minutes in increased β1-mediated adhesion to fibronectin and VCAM-1 at levels comparable to that obtained upon stimulation with the phorbol ester PMA. A role for PI 3-K in CD2-mediated increases in β1 integrin function is suggested by: (a) the ability of the PI 3-K inhibitor wortmannin to completely inhibit CD2-induced increases in β1 integrin activity; (b) the association of PI 3-K with CD2; and (c) induced PI 3-K activity upon CD2 stimulation. The mode of association of PI 3-K with CD2 is not mediated by tyrosine phosphorylation-dependent binding of PI 3-K via SH2 domains, since: (a) PI 3-K is associated with CD2 in unstimulated cells; (b) CD2 stimulation fails to increase the amount of associated PI 3-K; and (c) the CD2 cytoplasmic domain lacks tyrosine residues. A role for both protein kinase C and cytoskeletal rearrangements in CD2 regulation of integrin activity is also suggested, since a PKC inhibitor partially inhibits CD2- induced increases in β1 integrin function, and CD2 stimulation increases F- actin content in a wortmannin-sensitive manner. Analysis of human peripheral T cells indicated that CD2 stimulation also results in PI 3-K-dependent upregulation of β1 integrin activity. Thus, these results demonstrate that CD2 can function as an adhesion regulator in the absence of expression of the CD3/T cell receptor complex; and directly implicate PI 3-K as a critical intracellular mediator involved in the regulation of β1 integrin functional activity by the CD2 antigen.