The D-penicillamine autoimmune syndrome observed in Brown Norway (BN) rats is similar to an idiosyncratic reaction seen in some patients. We have previously shown that 2 weeks of low dose (5 mg/day) D-penicillamine pretreatment completely prevented all clinical signs of autoimmunity normally observed in 60-80% of rats treated with high dose (20 mg/day) D-penicillamine. We demonstrated that this tolerance is immune-mediated; tolerance to D-penicillamine is long lasting, we can adoptively transfer splenocytes from tolerant rats into slightly irradiated naive syngeneic recipients, and rats exposed to low dose D-penicillamine produce tolerogenic cytokines [Masson, M. J., and Uetrecht, J. P. (2004) Tolerance induced by low dose D-penicillamine in the brown norway rat model of drug-induced autoimmunity is immune-mediated. Chem. Res. Toxicol. 17, 82-94]. The aim of this study was to provide further understanding of the cells that are responsible for transferring tolerance and to assess the presence of regulatory T cells in the spleen of tolerant animals. We cotransferred T cells or splenocytes depleted of T cells from tolerant BN rats with splenocytes from naive BN rats into lightly irradiated syngeneic recipients. We found that neither tolerant splenocyte subpopulation could completely prevent clinical signs of autoimmunity. These results demonstrate that immune tolerance to D-penicillamine-induced autoimmunity may require both antigen presenting cells and T cells.