We have characterized the cellular immune response to human adenovirus 5 (Ad-5) in mice, as a basis for future study of responses to foreign antigens in recombinant adenoviruses. Primary in vivo lytic effector cells contained both virus immune cytolytic T (Tc) cells and natural killer cells. The Tc effectors could be boosted in vitro to give a secondary Tc cell response. The Tc cell response to Ad-5 was major histocompatibility complex (MHC) restricted, and in CBA/H (H-2k) mice mapped to the K end of MHC. Kinetic experiments suggested the Tc cell response was directed against early rather than late viral proteins. Experiments with viral mutants showed that the main responses were to the E1A and E1B proteins, with some involvement of E2. Expression of E3 and E4 in infected targets was not required, in fact lysis of target cells infected by viruses with a deletion in E3 was augmented.