The lytic function of human natural killer (NK) cells is markedly influenced by recognition of class I major histocompatibility complex (MHC) molecules, a process mediated by several types of activating and inhibitory receptors expressed on the NK cell. One of the most important of these mechanisms of regulation is the recognition of the non‐classical class I MHC molecule HLA‐E, in complex with nonamer peptides derived from the signal sequences of certain class I MHC molecules, by heterodimers of the C‐type lectin‐like proteins CD94 and NKG2. Using soluble, recombinant HLA‐E molecules assembled with peptides derived from different leader sequences and soluble CD94/NKG2‐A and CD94/NKG2‐C proteins, the binding of these receptor–ligand pairs has been analysed. We show first that these interactions have very fast association and dissociation rate constants, secondly, that the inhibitory CD94/NKG2‐A receptor has a higher binding affinity for HLA‐E than the activating CD94/NKG2‐C receptor and, finally, that recognition of HLA‐E by both CD94/NKG2‐A and CD94/NKG2‐C is peptide dependent. There appears to be a strong, direct correlation between the binding affinity of the peptide–HLA‐E complexes for the CD94/NKG2 receptors and the triggering of a response by the NK cell. These data may help to understand the balance of signals that control cytotoxicity by NK cells.