Parenteral injection of a lipopeptide containing a human leucocyte antigen (HLA)-A*0201-restricted cytotoxic T-lymphocyte (CTL) epitope from the human cytomegalovirus (HCMV) immunodominant matrix protein pp65 efficiently induces systemic CTL responses in HLA-A*0201 transgenic mice. In this study, we demonstrate that intranasal (i.n.) administration of this lipopeptide, covalently linked to a universal T helper (Th) epitope (PADRE), also induces potent systemic CTL responses. Immune responses were substantially reduced when the unlipidated peptide analogue was used (P<0.01). The induced CTL were CD8+, major histocompatibility complex (MHC) class I-restricted and CMV specific. Moreover, i.n. administration of this lipidated peptide elicited both systemic and local mucosal CD4+ T-cell proliferative responses, as well as antigen-specific delayed type hypersensitivity (DTH) immune responses. In contrast, mice receiving the unlipidated peptide analogue developed substantially reduced Th or DTH responses (P<0.05). These results highlight the usefulness and potential of lipopeptides delivered via mucosal routes as painless, safe, and non-invasive vaccines.