The adenovirus early region 3 glycoprotein E3-19k binds to and inhibits expression of class I major histocompatibility complex (MHC)-encoded molecules, which may help infected cells evade immune recognition. The role of specific regions of the class I MHC molecule in the interaction with E3-19k was evaluated using a series of HLA-A2.1-, HLA-A2 variant-, and HLA-B7-expressing cell lines. The monoclonal antibody (mAb) W6/32, which recognizes a monomorphic epitope on class I MHC molecules, readily co-immunoprecipitated E3-19k with HLA-A2.1 and 14 different HLA-A2 variant molecules that differ from HLA-A2.1 by single amino acid substitutions. Thus, no single residue tested in the regions of the class I MHC molecule that bind peptide or the T-cell receptor controls the binding to E3-19k. Additional immunoprecipitations performed with mAbs directed against well-defined epitopes on the surface of HLA-A2.1 revealed a dichotomy in the ability of the mAbs to co-immunoprecipitate HLA-A2.1 and E3-19k. The mAbs LGIII-220 (directed against the C-terminal end of the alpha 1-helix), CR11-351 (directed against the N-terminal end of the alpha 2-helix), and PA2.1 (directed against the middle of the alpha 2-helix and an underlying beta-loop) readily co-precipitated HLA-A2.1 and E3-19k. In contrast, mAbs MA2.1 (directed against the N-terminal end of the alpha 1-helix and the C-terminal end of the alpha 2-helix) and HO-2 (directed against the N-terminal end of the alpha 1-helix) did not co-precipitate E3-19k with HLA-A2.1. Similarly, mAb MB40.2 (directed against residues 169-182 of HLA-B7) also did not co-precipitate E3-19k with HLA-B7. These studies lead to the conclusion that the N-terminal end of the alpha 1-helix and the C-terminal end of the alpha 2-helix play an important role in dictating the ability of the E3-19k protein to bind to the class I MHC molecule.