To test whether simple expression units used in DNA vaccines can generate immunogenic, MHC class I-binding epitopes by translating other than the primary open reading frame (ORF), we constructed a vector (pCI/SX) that encodes the small hepatitis B surface Ag in the primary ORF, and a C-terminal fragment (residue 344-832) of the polymerase (Pol) in an alternative (out-of-frame) reading frame. pCI/SX efficiently primed multispecific, HLA-A2-restricted CD8+ T cell responses to epitopes of hepatitis B surface Ag and of Pol (Pol3, Pol(803-811)). Pol3-containing products generated from pCI/SX were detected only by T cell assays, but not by biochemical assays. Priming Pol-specific T cell responses to epitopes generated from alternative ORFs depended on promoter sequences that drive transcription in the DNA vaccine (human CMV-derived promoter sequences being more efficient than SV40-derived promoter sequences). Human CMV promoter-driven Pol constructs encoding different Pol fragments in primary or alternative reading frames elicited comparable levels of Pol3-specific T cell responses. We confirmed efficient T cell priming to epitopes from alternative ORFs by constructing DNA vaccines that encode an SV40-derived cT(1-272) protein fused either in frame or out of frame with an immunogenic OVA fragment (OVA(18-385)). Similar OVA-specific CD8+ T cell responses were primed by both alternative vaccine constructs. Hence, DNA vaccine-stimulated T cell responses to epitopes generated from alternative ORFs seem to be a regular event, although its biological role and risks are largely unexplored.