The glycoprotein B (gB) from herpes simplex virus type I is a major target of cytotoxic T lymphocytes (CTL) in C57BL/6 mice. The majority of these T cells are directed to a single Kb-restricted determinant, gB498-505. We have analyzed the T-cell receptor (TCR) usage in gB-specific CTL lines derived shortly after virus infection. The CTL populations preferentially used two V beta regions, a dominant V beta 10 element and a subdominant V beta 8 element. Detailed sequence analysis revealed considerable TCR beta-chain heterogeneity despite a striking level of predicted amino acid conservation at the V beta-D beta junction. This junction forms part of the third hypervariable loop of the TCR thought to directly contact the major histocompatibility complex-bound antigenic peptide. The results reveal considerable diversity within the primary T cells responding to a single viral determinant while still maintaining a high degree of TCR V beta bias and sequence conservation at the V-D-J junction.