Major histocompatibility complex class I–restricted cytotoxic T lymphocytes (CTLs) specific for epitopes within eight of the nine Epstein Barr Virus (EBV)-encoded latency-associated proteins have been recovered from EBV-infected human subjects by restimulation of lymphocytes in vitro. However, human class I–restricted CTL responses capable of recognizing EBNA-1 expressing cells were not detected in these studies. We have raised a murine CTL line that recognizes an epitope within EBNA-1 by immunizing mice with a vaccinia virus encoding a COOH-terminal EBNA-1 fragment. This novel CTL line was used to investigate whether the epitope (positions 509–517 in EBNA-1, presented through K^d) was presented to CTL by mouse cells expressing full-length EBNA-1 or a deletion mutant of EBNA-1, lacking the Glycine-Alanine (Gly-Ala)–rich region. Cells expressing full-length EBNA-1 are not lysed by the CTL line, whereas cells expressing the Gly-Ala deletion mutant are recognized. These results suggest that epitopes from full-length EBNA-1 are poorly presented, and that the Gly-Ala–rich region is responsible for this phenomenon. The inefficient presentation of EBNA-1–derived epitopes may explain the absence or rarity of EBNA-1–specific CTLs in vivo, a strategy that may allow EBV to maintain persistence within the immunocompetent host without being eliminated by CTLs.