We have previously shown that the immunogenicity of protein antigens can be significantly enhanced if electrostatically associated with the Toll-like receptor-2 agonist-based lipopeptide R4Pam2Cys. The precise mechanisms and effectiveness of the cytotoxic T-lymphocyte (CTL)-mediated response facilitated by this agonist, however, have not been studied. Here we show that priming by dendritic cells (DCs) in the draining lymph nodes of animals vaccinated with antigen delivered using R4Pam2Cys results in significantly improved T-cell proliferation and induces their differentiation into polyfunctional effector CTLs characterised by granzyme B expression and the ability to secrete interferon-γ, interleukin-2 and tumor necrosis factor-α 7 days after vaccination. After 30 days, frequencies of antigen-specific CD62(low)CD127(high) (effector memory), CD62(high)CD127(high) (central memory) and CD43(low)CD27(high) CD8(+) T cells, a phenotype associated with strong recall responses against respiratory infections, are also increased compared with responses obtained with antigens formulated in the adjuvants Alum (alhydrogel) and CFA (complete Freund's adjuvant). The phenotypic changes observed in these mice vaccinated using R4Pam2Cys further correlated with their ability to recall specific T cells into the lung to mediate the reduction of pulmonary viral titres following challenge with a chimeric influenza virus containing the K(b)OVA257-264 epitope compared with animals vaccinated using Alum or CFA. The findings from this study not only demonstrate that better T-cell responses can be elicited using R4Pam2Cys compared with classically utilised adjuvants but also highlight the potential effectiveness of this lipopeptide-based adjuvant particularly against viral infections that require resolution through cell-mediated immunity.Immunology and Cell Biology advance online publication, 7 January 2014; doi:10.1038/icb.2013.102.