Expression of adenovirus E1A gene products in tumor cells enhances NK cell lysis in vitro and NK-mediated rejection in vivo, despite increasing class I molecules on tumor cells. It is unclear why the increased expression of MHC class I molecules does not appear to confer resistance to killing by NK cells. One possibility is the unique capacity of E1A to sensitize cells to multiple NK cell killing mechanisms including perforin/granzyme, Fas ligand, tumor necrosis factor-alpha and TRAIL. To examine this issue, MCA-102-E1A tumor cells (H-2(b)) that express E1A and are NK sensitive were transfected with H-2D(d), the ligand for the NK inhibitory receptor, Ly49A. Expression of H-2D(d) molecules by MCA-102-E1A cells protected them from lysis by a Ly49A(+) NK cell clone and Ly49A(+) NK cells isolated from C57BL/6 nude mice. In contrast, NK cell-mediated rejection of MCA-102-E1A tumor cells was not inhibited by the expression of H-2D(d) molecules, nor was killing by polyclonal populations of NK cells isolated from C57BL/6-nude mice. H-2D(d) interacts with several inhibitory Ly49 receptors that are non-clonally expressed on NK cells in C57BL/6 mice: Ly49A (20% of NK cells), Ly49G2 (54% of NK cells) and Ly49C/I (47% of NK cells). Our data indicate that while E1A sensitizes cells to NK cell killing, it does not interfere with signal transduction by inhibitory NK receptors. Therefore, a small population of NK cells that do not express Ly49A, Ly49G2 or Ly49C/I inhibitory receptors are likely responsible for the rejection of MCA-102-E1A-D(d) tumor cells in vivo.