The E3/19K protein of human adenovirus type 2 binds to class I MHC Ags thereby interfering with their cell surface expression and Ag presentation function. Currently, it is unclear exactly which structure of MHC molecules is recognized by the E3/19K protein. We have previously demonstrated that the murine H-2Kd Ag is able to associate with E3/19K, whereas the allelic H-2Kk molecule is not. By using exon shuffling between Kd and Kk molecules, the alpha 1 and alpha 2 domains of MHC class I molecules were identified as essential structures for binding the viral protein. In this report, we have examined the contribution of individual amino acids within the alpha 2 domain of MHC for binding E3/19K. First, we show that within this domain the alpha-helical part is most important for the interaction with E3/19K. By using site-directed mutagenesis, Kd-specific amino acids were introduced into the alpha-helix of the alpha 2 domain of Kk. By using the expression of mutagenized proteins in E3/19K+ cells, we have identified Tyr 156 and Leu 180 as being essential for the association with the E3/19K protein. In addition, Kd residue Glu 163 seems to contribute to the complex formation. Furthermore, analysis of a panel of Kd/Dd recombinants indicates that a similar region in the Dd molecule, namely, the C-terminal half of the alpha 2 domain, affects binding to E3/19K. Combining these results with Ab binding data, we present two alternative models of how the adenovirus protein may bind to the alpha 1 and alpha 2 domains.