Dendritic cells (DCs) are capable of cross-presenting exogenous Ag to CD8+ CTLs. Detection of microbial products by Toll-like receptors (TLRs) leads to activation of DCs and subsequent orchestration of an adaptive immune response. We hypothesized that microbial TLR ligands could activate DCs to cross-present Ag to CTLs. Using DCs and CTLs in an in vitro cross-presentation system, we show that a subset of microbial TLR ligands, namely ligands of TLR3 (poly (inosinic-cytidylic) acid) and TLR9 (immunostimulatory CpG DNA), induces cross-presentation. In contrast to presentation of Ag to CD4+ T cells by immature DCs, TLR-induced cross-presentation is mediated by mature DCs, is independent of endosomal acidification, and relies on cytosolic Ag processing machinery.