Intraocular injection of exogenous protein induces an Ag-specific impairment of systemic delayed hypersensitivity (DH), termed anterior chamber associated immune deviation (ACAID). The ACAID-inducing signal is carried by blood-borne cells from the eye to the spleen and can also be generated in vitro by incubating APCs with Ag plus TGF-beta. Paradoxically, class I-restricted CD8 regulatory cells are induced in the spleens of mice with ACAID, and previous studies suggest that CD8 cells are important, and even necessary, for the expression of ACAID. To explore this issue further, we asked whether ACAID could be induced with a class II-restricted peptide, and if so, what type of regulatory cells are generated. An intraocular, but not an i.v., injection of OVA (323-339) peptide resulted in impairment of native OVA-specific DH in both naive and previously sensitized mice. Furthermore, i.v. injection of APCs pretreated with TGF-beta plus OVA peptide also prevented native OVA-specific DH. Surprisingly, both CD8+ and CD4+ spleen cells capable of impairing expression of DH were induced by either intraocular injection of peptide or i.v. injection of APCs pretreated with peptide plus TGF-beta. In summary, ACAID can be induced by a class II-restricted peptide and is accompanied by the generation of two unusual populations of cells: 1) CD8+ regulatory cells unexpectedly induced by class II-restricted peptide; and 2) a novel population of CD4 regulatory cells induced by peptide, but not native protein. Potential mechanisms involved in the processing and presentation of exogenous protein in the ACAID model are discussed in light of the present data.