The recognition of foreign antigen by T lymphocytes requires direct contact with cells expressing the antigen. It has recently become clear that T lymphocytes can form conjugates with other cells in the absence of foreign antigen expression. Studies using monoclonal antibodies (mAbs) to inhibit conjugate formation have suggested that a portion of the antigen-dependent adhesion is mediated by T lymphocytes interacting with cells expressing lymphocyte-function-associated antigen 3 (LFA-3), a widely distributed cell surface protein. We have investigated antigen-independent adhesion by incorporating affinity-purified LFA-3 into the lipid membrane of an artificial target cell (ATC; a nylon-matrix vesicle with a lipid membrane). These vesicles are similar in size and density to intact cells, so that conjugates between cells and ATCs may be seen by light microscopy. ATCs expressing a density of LFA-3 similar to that on intact cells were found to form conjugates with T cells, but only if the T cells expressed the sheep erythrocyte receptor, CD2 (T11; LFA-2). Previous studies using mAbs have implicated the CD2 molecule in both adhesion and T-cell activation. ATCs prepared without surface protein or with purified HLA class I protein failed to interact with the CD2-positive T cells, indicating that the adhesion found was mediated by the LFA-3 molecule. Furthermore, mAb against LFA-3 or CD2 was able to block the LFA-3-mediated vesicle-cell interaction, whereas mAb against LFA-1 or HLA failed to inhibit the interaction. These results provide direct evidence that LFA-3 functions as an adhesion molecule by serving as a ligand for the CD2 molecule on T cells.