Some HLA-C alleles have been shown to exert a specific protective effect preventing target cells from lysis by groups of natural killer (NK) clones displaying a defined specificity. In this study, we analyzed whether class I-mediated protection is a more general phenomenon involving all NK cells. First, we utilized two anti-class I mAbs (6A4 of IgG1 isotype and A6-136 of IgM isotype), which had been shown to induce lysis of protected target cells by group 1 and group 2 NK clones. Addition of A6-136 or 6A4 used as F(ab')2 mAb resulted in lysis of protected target cells by all NK clones analyzed. Target cells were represented by a panel of HLA homozygous Epstein-Barr virus-transformed B cell lines (B-EBV) while NK clones were representative of clones displaying different GL183/EB6 surface phenotypes and/or different abilities to lyse allogeneic cells. Unselected NK clones derived from seven different individuals were tested against autologous target cells represented by phytohemagglutinin-induced blasts or B-EBV transformed cell lines. In both instances, addition of a mixture of 6A4 F(ab')2 and A6-136 mAbs resulted in lysis of autologous target cells, thus suggesting that class I molecules prevent lysis of normal cells by self NK cells. We further investigated whether the class I-mediated protection requires the complexed form of class I molecules (composed of alpha chain, beta 2-microglobulin and the antigen peptide) or rather the free alpha chain. Acidic treatment of the C1R (Cw4+) target cells or 81.22 (Cw3+, Cw4+) at pH 2.2 resulted in loss of reactivity with 6A4, A6-136 and W6-32 mAb (known to react with the assembled form of class I molecules) and in the de novo reactivity with L31 mAb (specific for the HLA-C free chain). While the untreated Cw+ C1R cells were resistant to lysis by the Cw4-specific group 1 NK clones, the pH 2.2-treated cells became highly susceptible to lysis by the same clones. These data indicate that, at least for the NK clones analyzed, the protection of target cells requires class I molecules in the complexed form.