Toll-like receptors (TLRs) play a crucial role in the initiation of innate responses following microbial infection and also in adaptive immune responses by orchestrating the activation of different cell populations. TLRs are expressed at high levels in antigen-presenting cells and recent studies have demonstrated the expression and biological role of TLRs in mouse and human CD4⁺ T cells. In this study, we analyzed TLR mRNA expression in rat CD4⁺ T cells using stringent quantitative reverse transcription–PCR conditions enabling a direct comparison of the levels of each TLR. We show that TLR3, 5, 6 and 9 mRNAs are the highest TLRs expressed in rat CD4⁺ T cells and that TLR5 mRNA is highly expressed in regulatory CD4⁺ CD25⁺ T cells. In addition, we show that the TLR9 ligand (TLR9L), CpG oligodeoxynucleotide, synergizes with anti-CD3 to induce proliferation of both CD4⁺ CD25⁻ and regulatory CD4⁺ CD25⁺ T cells and that TLR9L partially abrogates the suppressive activity mediated by regulatory CD4⁺ CD25⁺ T cells. This loss of suppression is in part due to the direct effect of TLR9L on effector T cells that are rendered more resistant to the regulation exerted by regulatory T cells. To our knowledge, this is the first study describing the expression of TLR mRNA in rat CD4⁺ T cells and the capacity of TLR9L to directly regulate rat T cell responses. Thus, TLR9L may rapidly increase the host's adaptive immunity by expanding effector cells and also by attenuating the suppressive activity mediated by regulatory T cells.