The CD8⁺-T-cell response to Moloney murine leukemia virus (M-MuLV)-associated antigens in C57BL/6 mice is directed against an immunodominant gag-encoded epitope (CCLCLTVFL) presented in the context of H-2D^b and is restricted primarily to cytotoxic T lymphocytes (CTL) expressing the V3.2 and V5.2 gene segments. We decided to examine the M-MuLV response in congenic C57BL/6 V^a mice which are unable to express the dominant V3.2⁺ V5.2⁺ T-cell receptor (TCR) due to a large deletion at the TCR locus that includes the V5.2 gene segment. Interestingly, M-MuLV-immune C57BL/6 V^a mice were still able to reject M-MuLV-infected tumor cells and direct ex vivo analysis of peripheral blood lymphocytes from these immune mice revealed a dramatic increase in CD8⁺ cells utilizing the same V3.2 gene segment in association with two different V segments (V3 and V17). Surprisingly, all these CTL recognized the same immunodominant M-MuLV gag epitope. Analysis of the TCR repertoire of individual M-MuLV-immune (C57BL/6 × C57BL/6 V^a)F₁ mice revealed a clear hierarchy in V utilization, with a preferential usage of the V17 gene segment, whereas V3 and especially V5.2 were used to much lesser extents. Sequencing of TCR- and --chain junctional regions of CTL clones specific for the M-MuLV gag epitope revealed a diverse repertoire of TCR chains in V^a mice and a highly restricted TCR-chain repertoire in V^b mice, whereas TCR-chain sequences were highly conserved in both cases. Collectively, our data indicate that the H-2D^b-restricted M-MuLV gag epitope can be recognized in a hierarchal fashion by different V domains and that the degree of -chain diversity varies according to V utilization.