Cell-cell adhesion is essential for many immunological functions, including interaction of cytotoxic T lymphocytes (CTLs) with their targets. We have explored CTL-target interactions using well-characterized cloned human CTLs. Conjugate formation between these CTLs and many antigen-negative targets is almost as efficient as with specific target cells, but does not lead to target-cell lysis. Thus, on specific target cells, adhesion by antigen-independent pathways may occur concurrently with or precede antigen recognition. The molecules LFA-1, CD2 (T11, LFA-2) and LFA-3 have been shown to be involved in human CTL conjugation with and lysis of specific target cells. Here we describe monoclonal antibody inhibition studies using individual monoclonal antibodies and mixes which demonstrate (1) that LFA-1, CD2 and LFA-3 are involved in antigen-independent conjugate formation; and (2) suggest that CD2 and LFA-3 are involved in one pathway and LFA-1 in another. We confirmed the existence of distinct pathways by the demonstration that LFA-1-dependent adhesion requires divalent cations and is temperature-sensitive whereas CD2- and LFA-3-dependent adhesion does not require divalent cations and is temperature-insensitive. Together with previous data, our studies suggest that CD2 on the effector interacts with LFA-3 as its ligand on targets.