We explored the topical use of resiquimod (R-848), a Toll-like receptor (TLR) 7/8 agonist, in gel formulation, to enhance cross-priming to subcutaneously administered protein antigen in a murine model. Resiquimod application at the time of subcutaneous administration of ovalbumin generated robust antigen-specific CTL as detected by tetramers, IFN-gamma ELISPOT assays and standard cytotoxicity assays. Induced CTL were capable of mediating antigen-specific killing in vivo as measured by in vivo cytotoxicity assays and an ability to protect against B16-OVA tumor challenge. Multiple serial applications of topical resiquimod increased the frequency of antigen-specific CTL when compared to single application. This enhanced frequency was noted despite a marked inhibition of adjuvant mediated pro-inflammatory cytokine release following repeated administration. Topical resiquimod is a potent adjuvant for locally administered subcutaneous vaccines, inducing clinically relevant CTL responses following single application at the time of subcutaneous vaccination.