Friend virus infection of mice induces the expansion and activation of regulatory T cells (Tregs) that dampen acute immune responses and promote the establishment and maintenance of chronic infection. Adoptive transfer experiments and the expression of neuropilin-1 indicate that these cells are predominantly natural Tregs rather than virus-specific conventional CD4(+) T cells that converted into induced Tregs. Analysis of Treg TCR Vβ chain usage revealed a broadly distributed polyclonal response with a high proportionate expansion of the Vβ5(+) Treg subset, which is known to be responsive to endogenous retrovirus-encoded superantigens. In contrast to the major population of Tregs, the Vβ5(+) subset expressed markers of terminally differentiated effector cells, and their expansion was associated with the level of the antiviral CD8(+) T cell response rather than the level of Friend virus infection. Surprisingly, the expansion and accumulation of the Vβ5(+) Tregs was IL-2 independent but dependent on TNF-α. These experiments reveal a subset-specific Treg induction by a new pathway.