Mouse splenic dendritic cell (DC) subsets possess distinct antigen-presentation abilities. CD8(+) DC are specialized in cross-presentation of antigens to CD8(+) T cells, whereas CD8(-) DC are more efficient in antigen presentation to CD4(+) T cells. In this study, we examined the capacity of CD8(+) and CD8(-) DC subsets to present fungal antigens in MHC class I and II molecules to CD8(+) and CD4(+) T cells, respectively. We used ovalbumin-expressing Saccharomyces cerevisiae (yeast-OVA) as a fungal model system. Both CD8(+) and CD8(-) DC subsets phagocytosed yeast in equal amounts and uptake was mediated via dectin-1. In addition, both DC subsets induced similar OVA-specific CD4(+) T cell proliferation after incubation with yeast-OVA. However, the induction of OVA-specific CD8(+) T cell activation was largely restricted to the CD8(-) DC subset. Furthermore, only CD8(-) DC produced cytokines such as IL-10 and TNF-alpha and increased IL-23p19 and IL-23p40 mRNA levels in response to yeast. Our results strongly suggest that DC subsets have different functions in the elicitation of adaptive immune responses in vivo.