In the present study a unique antibody (NKI-L16) reacting with the alpha-chain of the human leukocyte function-associated Ag-1 (LFA-1) is described, which stimulates homotypic cell-cell interactions in a manner very similar to 12-O-tetradecanoyl-phorbol-13-acetate (TPA), in contrast to other anti-LFA-1 mAb which inhibit cell aggregation. The induction of aggregate formation of EBV-transformed B cells (JY) and CTL clones by TPA or NKI-L16 is not accompanied by an increase in the expression of LFA-1. Nevertheless, this cluster formation is LFA-1 dependent, inasmuch as anti-LFA-1 antibodies, other than NKI-L16, completely abrogate aggregation. Simultaneous addition of NKI-L16 and TPA did not result in a further increase of the speed of cluster formation, suggesting that a similar pathway is activated. Immunoprecipitation and enzyme digestion studies revealed that NKI-L16 recognizes a unique epitope on the alpha-chain of LFA-1, most likely situated close to the transmembrane segment of the molecule. It is hypothesized that NKI-L16 or TPA can cause the LFA-1 molecule to convert from an inactive to an active configuration, thereby permitting binding of LFA-1 to its natural ligand.