The injection of antigen into the anterior chamber (AC) induces the production of antigen-specific splenic CD8+ regulatory T cells (Tregs) /suppressor T cells that perform the local suppression of delayed-type hypersensitivity (DTH) responses. Because CD94/NKG2A-Qa-1-dependent interactions have been implicated in CD8+ Treg-mediated immune suppression and DBA/2J mice are deficient in CD94/NKG2R, we have utilized these mice to test the hypothesis that the CD94/NKG2A-Qa-1 system is essential to the induction and immunosuppressive function of CD8+ Tregs in anterior chamber-associated immune deviation (ACAID). We show that: (i) neither ACAID-mediated suppression of DTH to ovalbumin nor splenic Tregs/suppressor T cells was induced in DBA/2J mice that received an injection of antigen into the AC; (ii) splenic CD8+ Tregs from ACAID-induced DBA/2NCr mice suppressed the initiation of DTH when transferred to DBA/2J mice; (iii) following injection of antigen into the AC, intravenous administration of splenocytes or Peripheral Blood Mononuclear Cells (PBMC) isolated from DBA/2NCr but not from DBA/2J mice transferred suppression of DTH to DBA/2NCr mice; (iv) antibodies to CD94/NKG2A reduced the ACAID CD8+ T cell-mediated suppression of DTH and (v) The deficiency of such immune regulation in DBA/2J mice also correlated with a decreased number of Qa-1(b+) B cells, F4/80+ cells, a deficient number of CD94/NKG2AR and Qa-1 tetramer binding by CD8+ T cells. These results demonstrate that defective ACAID in DBA/2J mice involves multiple regulatory lesions resulting in a lack of induction of a CD8+ Treg response and possibly defective CD94/NKG2A-dependent suppression of peripheral cell-mediated immunity.