Topical application of adjuvant to the skin promotes the generation of immune responses to co-administered peptide or protein antigen. We demonstrate that topical administration of CpG adjuvant (a TLR9 agonist) induces the cross-presentation of, and antigen-specific CTL induction to, locally injected soluble protein antigen. C57BL/6 mice were immunized by subcutaneous or intramuscular injection with ovalbumin (OVA) protein as model antigen. Application of CpG to the local skin induced more efficient cross-presentation of the injected antigen than co-injected adjuvant. Robust antigen-specific CTL responses were generated, as determined by antigen-specific CTL enumeration using tetramers, IFN-gamma ELISPOT analysis and cytotoxicity assays. Long-term memory CTL responses were induced. Topical administration of adjuvant induced Langerhans cell migration, local type 1 IFN-dependent myxovirus-resistance protein A expression and bystander dendritic cell (DC) activation. Soluble antigen-bearing DC within the skin draining lymph nodes were mainly CD11chiCD11bhilangerinloDEC205lo. Topical administration did not result in the splenomegaly or systemic cytokine induction (including TNF-alpha, IL-12, IFN-gamma and MCP-1) noted with parenteral administration. Topical TLR9 family agonists may be used to modulate the immune response to soluble protein vaccines administered by standard percutaneous route. Topical adjuvant administration increases efficacy of CTL induction and reduces toxicity when compared to parenteral adjuvant administration.