To map and characterize functional differences between the E1A oncoproteins of Ad5 and Ad12, we previously constructed a series of hybrid Ad5/12 E1A genes and used them in combination with Ad12 E1B to transform Hooded Lister rat cells. At least two regions within the first exon of Ad12 E1A which influenced tumorigenicity were identified. In this report, again using the hybrid Ad5/12 E1A (plus Ad12 E1B) transformants, we further examined the role of these regions in tumorigenicity by analyzing their effect on cell surface major histocompatibility complex class I expression and sensitivity to class I-restricted CD8+ cytotoxic T lymphocytes (CTLs). Results of these studies suggest that expression of either of the Ad12 E1A regions implicated in tumorigenicity could down-regulate cell surface class I levels. However, neither class I down-regulation nor sensitivity to allogeneic CTLs was shown to strictly correlate with the tumorigenic capacities of the transformed rat cells. Another factor influencing the tumorigenicity of Ad5 E1 and Ad12 E1 transformants may be the ability of their E1A products to encode CTL epitopes. To this end, we provide evidence suggesting that CTL epitopes may be encoded by Ad5 E1A but not by Ad12 E1A, since expression of certain portions of the Ad5 E1A protein conferred susceptibility to syngeneic Ad5 E1-specific CTLs in vitro, while Ad12 E1A expression did not confer susceptibility to syngeneic Ad12 E1-specific CTLs.