Plastic adherent interleukin-2-activated human natural killer (NK) cells (ALAK) lyse many different histological types of tumor target cells. In order to effect their function as cytotoxic mediators of innate immunity, ALAKs may 'recognize' antigen(s) of protozoan parasites, select virus-infected cells and they may release certain cytokines in response to bacterial antigens. In the present study, we demonstrate that CD3-/CD56dim/CD16dim/monoclonal antibody 5C6bright human ALAKs bind to an antigenic determinant on tumor cells independent of target cell H-2 allotype expression. The conserved antigen was originally obtained from the protozoan Tetrahymena pyriformis, however it is also located on the membranes of many ALAK-sensitive tumor cells. The sequence of this protein, i.e. NK target antigen/NKTag, was previously deduced from cDNA. One ALAK cognate determinant of NKTag was identified by inhibition of cytotoxicity using NKTag-derived synthetic peptides. Biotinylated synthetic peptide [amino acids (aa) 58-74] bound to ALAKs, and synthetic peptides corresponding to this sequence inhibited ALAK lysis of U937 target cells. Inhibition effects of peptide binding were nonreversible. To determine the requirements for recognition by ALAKs of this antigenic determinant, the cognate peptide aa 55-74 was truncated to 17-, 14-, 10-, 7- and 6-mer lengths and tested for inhibition of cytotoxicity. All inhibited except the 6-mer. A possible mechanism of peptide inhibition of cytotoxicity following ALAK binding to an antigenic determinant was a requirement for recognition of one anchor peptide (arginine) and receptor occupancy by a minimum of five to six additional amino acids. In antibody-dependent cell-mediated cytotoxicity experiments, synthetic peptide (aa 68-74) inhibited ALAK killing of anti-H-2d-sensitized P815 targets. This same peptide also inhibited conventional lysis of nonsensitized P815 and IM-9 targets. However, the cognate synthetic peptide (aa 58-74) did not inhibit conjugate formation between ALAKs and U937 target cells. These data demonstrate that ALAK binding to a soluble monomeric peptide inhibited cytotoxicity. Peptide binding appeared to negatively regulate cytotoxicity, and the inhibitory effects following peptide binding were nonreversible. Effector:target cell conjugate formation was not affected by peptide binding, however, recognition was required because inhibition was specific for the amino acid sequence of the synthetic peptide.