NK cells are regulated by inhibiting and activating cell surface receptors. Most inhibitory receptors recognize MHC class I Ags and protect healthy cells from NK cell–mediated autoaggression. However, certain activating receptors, including the human activating killer cell Ig-like receptor (KIR) 2DS1, also recognize MHC class I. This fact raises the question of how NK cells expressing such activating receptors are tolerized to host tissues. We investigated whether the presence of HLA-C2, the cognate ligand for 2DS1, induces tolerance in 2DS1-expressing NK cells. Anti–HLA-C2 activity could be detected in vitro in some 2DS1 positive NK clones irrespective of the presence or absence of HLA-C2 ligand in the donor. The frequency of anti–HLA-C2 reactivity was high in donors homozygous for HLA-C1. Surprisingly, no significant difference was seen in the frequency of anti–HLA-C2 cytotoxicity in donors heterozygous for HLA-C2 and donors without HLA-C2 ligand. However, donors homozygous for HLA-C2, compared with all other donors, had significantly reduced frequency of anti–HLA-C2 reactive clones. The 2DS1 positive clones that express inhibitory KIR for self–HLA class I were commonly noncytotoxic, and anti–HLA-C2 cytotoxicity was nearly exclusively restricted to 2DS1 single positive clones lacking inhibitory KIR. 2DS1 single positive NK clones with anti–HLA-C2 reactivity were also present posttransplantation in HLA-C2 positive recipients of hematopoietic stem cell transplants from 2DS1 positive donors. These results demonstrate that many NK cells with anti–HLA-C2 reactivity are present in HLA-C1 homozygous and heterozygous donors with 2DS1. In contrast, 2DS1 positive clones from HLA-C2 homozygous donors are frequently tolerant to HLA-C2.