T cell epitopes coupled to a lipid moiety (lipopeptides) may be superior immunostimulants compared to peptide antigens and are currently studied as potential vaccines. The cause of enhanced immunogenicity of lipopeptides is largely unknown but members of the novel family of Toll like receptors (TLR) such as TLR2 and TLR4 have been shown to mediate activation of cells in response to bacterial lipopolysaccharide (LPS) and other lipidated bacterial or viral components. We studied TLR-mediated activation by 14 synthetic lipopeptides corresponding to T cell epitopes on hepatitis C virus (HCV) core in human embryonic kidney cells (HEK293) transiently over-expressing TLR2 and in Ba/F3 mouse bone marrow cells stably transfected with TLR4 and the adaptor molecule MD-2. Stimulation of transfected HEK293 or Ba/F3 cells was measured via luciferase activity as a reporter of nuclear factor kappaB activation. Free peptides, a non-HCV-related lipopeptide as well as LPS and the lipopeptide SK4 were used as controls. Ten of the 14 HCV core lipopeptides stimulated luciferase activity in TLR2-transfected HEK293 cells but not in mock-transfected control cells. Nine of the 14 lipopeptides also stimulated luciferase activity in the TLR4/MD-2 double-transfected Ba/F3 cells but not Ba/F3 control cells. Overall, there was a close statistical correlation between TLR2 and TLR4/MD-2-mediated cell activation by the lipopeptides. In contrast, the corresponding free peptides had no stimulatory effect on TLR2 nor on TLR4/MD-2 transfected cells. Thus, lipopeptides but not their corresponding free peptides can activate cells via TLRs 2 and 4. This activation is apparently affected by the amino acid sequence of the peptide moiety.