Abstract Target cell expression of certain MHC class I molecules correlates with resistance to lysis by NK cells. To explain this correlation, one hypothesis states that NK cells may possess two types of receptors; one may activate NK cells whereas another, specific for target cell MHC class I molecules, may inhibit natural killing by transducing negative signals. The cell surface molecule, Ly-49, is expressed on an NK cell subpopulation (15% to 20%) in spleens from C57BL/6 (H-2b) mice. Previously, we showed that lysis by Ly-49+ IL-2-activated NK cells was globally inhibited when targets expressed either H-2Dd or an H-2k-class I molecule, consistent with the hypothesis that Ly-49 is an inhibitory NK cell receptor that engages these MHC class I molecules. We now have determined the influence of specific host MHC class I molecules on Ly-49 expression. In two-color flow cytometric examination of splenic cells, Ly-49+ NK1.1+ cells were undetectable in MHC-congenic strains expressing Dd or Dk, in C57BL/6 mice transgenic for membrane-bound Dd, and in B10.D2dm1 mice. These data establish that Dd itself is sufficient for this effect and suggest that Ly-49 engages Dd-alpha 1/alpha 2 domains. Cross-linking of Ly-49 with membrane-bound Dd may be required because Ly-49+ NK1.1+ cells were readily detectable in C57BL/6 strains transgenic for soluble forms of Dd. To examine whether this effect could be the result of down-regulation of Ly-49 expression or negative selection of Ly-49+ cells, we determined Ly-49 expression on highly purified, freshly isolated NK cell populations (> 90% NK1.1+ CD3- cells). These experiments demonstrated that Ly-49+ cells were present in normal numbers but that Ly-49 expression was markedly decreased in congenic mice expressing H-2Dd or Dk, and in the strain transgenic for membrane-bound H-2Dd. Thus, expression of a putative MHC class I-specific NK cell receptor is modulated by its apparent interaction with alpha 1/alpha 2 domains of host MHC class I molecules.