The mouse inhibitory NK cell receptor Ly49A recognizes the mouse MHC class I molecule H-2D(k). The present study focuses on the species specificity of beta(2)-microglobulin (beta(2)m), an invariant component of MHC class I, in the interaction between Ly49A and H-2D(k). Transfection of the beta(2)m-defective mouse cell line R1E/TL8x.1 with human (h) beta(2)m induced cell-surface expression of H-2D(k), but failed to protect the cells from killing by Ly49A(+) NK cells. In contrast, the cells transfected with mouse (m) beta(2)m were protected from killing by Ly49A(+) NK cells. These data indicate that Ly49A distinguishes mbeta(2)m from hbeta(2)m when it recognizes the H-2D(k) complexes. To identify the species-specific determinant of beta(2)m required for Ly49A recognition of H-2D(k), we prepared a panel of mbeta(2)m mutants and tested the H-2D(k) that included each of the beta(2)m mutants for its capacity to engage Ly49A on NK cells. Ly49A failed to functionally recognize the H-2D(k) that included the mbeta(2)m with K3R and Q29G mutations. Moreover, Ly49A was able to recognize the H-2D(k) that included the hbeta(2)m with R3K and G29Q mutations. These data indicate that Lys3 and Gln29 consist of the central part of the species-specific determinant of beta(2)m required for Ly49A recognition of H-2D(k). The two residues are conserved in the mouse and the rat, in which NK cells use Ly49 family molecules as the receptors specific for MHC class I. These results suggest functional importance of beta(2)m in NK cell recognition of target cells.