Cytotoxic T-lymphocyte (CTL) responses induced by persistent Epstein-Barr virus (EBV) infection in normal B-lymphoid tissues could potentially be directed against EBV-positive malignancies if expression of the relevant viral target proteins is maintained in tumor cells. For malignancies such as nasopharyngeal carcinoma and Hodgkin's disease, this will require CTL targeting against the nuclear antigen EBNA1 or the latent membrane proteins LMP1 and LMP2. Here we analyze in detail a B95.8 EBV-reactivated CTL response which is specific for LMP2 and restricted through a common HLA allele, A2.1. We found that in vitro-reactivated CTL preparations from several A2.1-positive virus-immune donors contained detectable reactivity against A2.1-bearing target cells expressing either LMP2A or the smaller LMP2B protein from recombinant vaccinia virus vectors. Peptide sensitization experiments then mapped the A2.1-restricted response to a single epitope, the nonamer CLGGLLTMV (LMP2A residues 426 to 434), whose sequence accords well with the proposed peptide binding motif for A2.1. Most Caucasian and African virus isolates (whether of type 1 or type 2) were identical in sequence to B95.8 across this LMP2 epitope region, although 2 of 12 such isolates encoded a Leu-->Ile change at epitope position 6. In contrast, most Southeast Asian and New Guinean isolates (whether of type 1 or type 2) constituted a different virus group with a Cys-->Ser mutation at epitope position 1. CTLs raised against the B95.8-encoded epitope were nevertheless able to recognize these variant epitope sequences in the context of A2.1 whether they were provided exogenously as synthetic peptides or generated endogenously in B cells transformed with the variant viruses. A CTL response of this kind could have therapeutic potential in that it is directed against a protein expressed in many EBV-positive malignancies, is reactive across a range of virus isolates, and is restricted through a relatively common HLA allele.