Successful establishment and persistence of adenovirus (Ad) infections are facilitated by immunosubversive functions encoded in the early transcription unit 3 (E3). The E3/19K protein has a dual role, preventing cell surface transport of MHC class I/HLA class I (MHC-I/HLA-I) Ags and the MHC-I-like molecules (MHC-I chain-related chain A and B [MICA/B]), thereby inhibiting both recognition by CD8 T cells and NK cells. Although some crucial functional elements in E3/19K have been identified, a systematic analysis of the functional importance of individual amino acids is missing. We now have substituted alanine for each of 21 aas in the luminal domain of Ad2 E3/19K conserved among Ads and investigated the effects on HLA-I downregulation by coimmunoprecipitation, pulse-chase analysis, and/or flow cytometry. Potential structural alterations were monitored using conformation-dependent E3/19K-specific mAbs. The results revealed that only a small number of mutations abrogated HLA-I complex formation (e.g., substitutions W52, M87, and W96). Mutants M87 and W96 were particularly interesting as they exhibited only minimal structural changes suggesting that these amino acids make direct contacts with HLA-I. The considerable number of substitutions with little functional defects implied that E3/19K may have additional cellular target molecules. Indeed, when assessing MICA/B cell-surface expression we found that mutation of T14 and M82 selectively compromised MICA/B downregulation with essentially no effect on HLA-I modulation. In general, downregulation of HLA-I was more severely affected than that of MICA/B; for example, substitutions W52, M87, and W96 essentially abrogated HLA-I modulation while largely retaining the ability to sequester MICA/B. Thus, distinct conserved amino acids seem preferentially important for a particular functional activity of E3/19K.