Downregulation of MHC class Ia molecule expression is a widespread mechanism used by tumor cells to escape antitumor T-cell-mediated immune responses. However, it is not known why NK cells cannot lyse these MHC class-Ia-deficient tumor targets. Tumors must select additional routes of escape from NK cells. An attractive hypothesis is that the aberrant expression of nonclassical HLA class Ia molecules in tumors provides the required inhibitory signal to NK cells, rendering tumor cells resistant to NK lysis. To analyze the possible role of HLA-E molecules in providing tumor cells with an NK escape mechanism, we studied the cell surface expression of this HLA class Ib molecule in a variety of tumor cell lines with well-defined HLA class Ia alterations. Tests were done with the monoclonal antibody 3D12 recognizing cell surface HLA-E molecules. Our results indicate that HLA-E was mainly detected in leukemia-derived cell lines. In addition, HLA-E was detected in tumor cell lines of different origin. This expression was related with the availability of free β₂-microglobulin (β₂m) in the cytoplasm of tumor cells. Expression was detected in tumor cell lines showing an imbalance in heavy chain/β₂m expression, particularly in tumor cell lines with alterations in the expression of heavy-chain genes. Several lines of evidence favor these conclusions: (1) In the FM55 and NW145 melanoma tumor systems, the reduction in HLA class Ia expression paralleled the increased cell surface detection of HLA-E. (2) A cervical tumor (808) and a melanoma cell line (R22.2) expressing a single HLA-A1 allele also expressed HLA-E. (3) The addition of human β₂m to tumor cell lines that expressed the HLA-E ^G allele increased HLA-E cell surface expression. (4) There was no HLA-E cell surface expression in tumor cell lines with total loss of HLA class Ia expression, including cell lines with low transcription of HLA class I heavy chains or with β₂m mutations. Our findings suggest that the biological consequences of these cumulative genetic and molecular changes in tumor cells lead to the appearance of HLA-E in a limited number of tumor cell lines with peculiar phenotypic and genotypic characteristics, namely: HLA-class Ia downregulation, free β₂m and HLA-E ^G genotype. The aberrant HLA-E expression might be of particular biological relevance in those HLA tumor phenotypes that express a single HLA-A allele when NK inhibition is markedly reduced due to the downregulation of HLA-B and -C alleles.