NKG2D is an activating receptor that is expressed on most natural killer (NK) cells, CD8 αβ T cells, and γδ T cells. Among its ligands is the distant major histocompatibility complex class I homolog MICA, which has no function in antigen presentation but is induced by cellular stress. To extend previous functional evidence, the NKG2D-MICA interaction was studied in isolation. NKG2D homodimers formed stable complexes with monomeric MICA in solution, demonstrating that no other components were required to facilitate this interaction. MICA glycosylation was not essential but enhanced complex formation. Soluble NKG2D also bound to cell surface MICB, which has structural and functional properties similar to those of MICA. Moreover, NKG2D stably interacted with surface molecules encoded by three newly identified cDNA sequences (N2DL-1, -2, and -3), which are identical to the human ULBP proteins and may represent homologs of the mouse retinoic acid-early inducible family of NKG2D ligands. Because of the substantial sequence divergence among these molecules, these results indicated promiscuous modes of receptor binding. Comparison of allelic variants of MICA revealed large differences in NKG2D binding that were associated with a single amino acid substitution at position 129 in the α2 domain. Varying affinities of MICA alleles for NKG2D may affect thresholds of NK-cell triggering and T-cell modulation.