The effect of dendritic cell (DC) maturation on MHC class II-restricted Ag presentation is well studied, but less is known about the effects of DC maturation on MHC class I-restricted cross-presentation. We investigated the ability of mature DCs to present Ags from cells infected with HSV-1. Pretreatment with pure LPS increased cross-presentation in a manner dependent on both MyD88 and Toll/IL-1R domain-containing adaptor inducing IFN-β, whereas a similar dose of a less pure LPS preparation inhibited cross-presentation. The difference could not be attributed to differences in uptake or phenotypic maturation. The likely contaminant responsible for shutting down cross-presentation is peptidoglycan (PGN). Addition of PGN to pure LPS abrogated its ability to enhance cross-presentation. Direct activation of DCs with PGN inhibited cross-presentation through nucleotide-binding oligomerization domain-like receptor signaling. These results demonstrate that different maturation stimuli can have opposite impacts on the ability of DCs to cross-present viral Ags.