The human Ag-processing-defective cell line 721.174, and its derivative T2, have a large homozygous deletion in the MHC class II region encompassing the TAP1 and TAP2 as well as the LMP2 and LMP7 genes. A similar, but smaller defect encompassing the TAP1 gene is observed in the 721.134 mutant cell line. With a few recent exceptions, these lines are unable to present endogenous Ag to class I-restricted CD8+ CTL. However, restoration of Ag processing and presentation to CD8+ cytotoxic T cells can be achieved by transfecting TAP1 and TAP2 genes into the T2 and .174 cells, or the TAP1 gene into .134 cells. In contrast to their resistance to MHC class I-restricted CTL-mediated lysis, T2 and .174 as well as .134 cells are highly sensitive to lysis by human NK cells. In the present study, we demonstrate the reversal of the NK-sensitive phenotype of these mutant cell lines upon transfection of TAP genes. The induced NK cell-resistant phenotype, studied in detail with TAP1/2-transfected T2 cells, appeared independent of the MHC class I haplotype of the NK cell donors and was also observed with xenogeneic NK cells. The resistance to NK cell lysis induced by re-expression of TAP1/2 seemed to be independent of the origin of the TAP genes; both human and rat transporters efficiently conferred NK cell resistance. Transfection of single TAP genes to T2 or .174 cells, whether TAP1 or TAP2, did not markedly affect NK cell susceptibility. This indicates that an intact TAP1/2 dimer, and thus efficient peptide translocation into the ER, is necessary for rendering T2 and .174, as well as .134 cells, resistant to NK cell-mediated lysis. Proper translocation of peptides across the ER membrane and loading to class I molecules may represent critical events in rendering an NK-resistant phenotype.