In vivo priming of CD8+ T lymphocytes against exogenously processed model Ags requires CD4+ T cell help, specifically interactions between CD40 ligand (CD40L) expressed by activated CD4+ T cells and CD40, which is present on professional APC such as dendritic cells (DCs). To address this issue in the context of bacterial infection, we examined CD40L-CD40 interactions in CD8+ T cell priming against an exogenously processed, nonsecreted bacterial Ag. CD40L interactions were blocked by in vivo treatment with anti-CD40L mAb MR-1, which inhibited germinal center formation and CD8+ T cell cross-priming against an exogenous model Ag, OVA. In contrast, MR-1 treatment did not interfere with CD8+ T cell priming against a nonsecreted or secreted recombinant Ag expressed by Listeria monocytogenes. Memory and secondary responses of CD8+ T cells against nonsecreted and secreted bacterial Ags were also largely unimpaired by transient MR-1 treatment. When MR-1-treated mice were concurrently immunized with L. monocytogenes and OVA-loaded splenocytes, cross-priming of OVA-specific naive CD8+ T cells occurred. No significant decline in cross-priming against OVA was measured when either TNF or IFN-γ was neutralized in L. monocytogenes-infected animals, demonstrating that multiple signals exist to overcome CD40L blockade of CD8+ T cell cross-priming during bacterial infection. These data support a model in which DCs can be stimulated in vivo through signals other than CD40, becoming APC that can effectively stimulate CD8+ T cell responses against exogenous Ags during infection.