Signalling through Toll-like receptors (TLRs) may play a role in the pathogenesis of autoimmune diseases, such as multiple sclerosis (MS). In the present study, the expression of TLR-2, -4 and -9 was significantly higher on CD4⁺ and CD8⁺ T-cells from MS patients compared to healthy individuals. Following in-vitro activation, the proportion of interleukin (IL)-17⁺ and IL-6⁺ CD4⁺ and CD8⁺ T-cells was higher in the patients. In addition, the proportion of IFN-γ-secreting TLR⁺ CD8⁺ T-cells was increased in MS patients. Among different IL-17⁺ T-cell phenotypes, the proportion of IL-17⁺ TLR⁺ CD4⁺ and CD8⁺ T-cells producing IFN-γ or IL-6 were positively associated with the number of active brain lesions and neurological disabilities. Interestingly, activation of purified CD4⁺ and CD8⁺ T-cells with ligands for TLR-2 (Pam3Csk4), TLR-4 [lipopolysaccharide (LPS)] and TLR-9 [oligodeoxynucleotide (ODN)] directly induced cytokine production in MS patients. Among the pathogen-associated molecular patterns (PAMPs), Pam3Csk4 was more potent than other TLR ligands in inducing the production of all proinflammatory cytokines. Furthermore, IL-6, IFN-γ, IL-17 and granulocyte-macrophage colony-stimulating factor (GM-CSF) levels produced by Pam3Csk4-activated CD4⁺ cells were directly associated with disease activity. A similar correlation was observed with regard to IL-17 levels released by Pam3Csk4-stimulated CD8⁺ T-cells and clinical parameters. In conclusion, our data suggest that the expansion of different T helper type 17 (Th17) phenotypes expressing TLR-2, -4 and -9 is associated with MS disease activity, and reveals a preferential ability of TLR-2 ligand in directly inducing the production of cytokines related to brains lesions and neurological disabilities.