Previous studies suggest that altered virus-specific T-cell responses observed during chronic ethanol exposure may be due to abnormal functioning of dendritic cells (DCs). Here we explored the effects of ethanol on exogenous antigen presentation by DCs. BALB/c, C57BL/6, and CBA/caj mice were fed ethanol or an isocaloric control diet for 8 weeks. The splenic DC population was expanded using an Flt3L expression plasmid via tail vein injection. DCs were purified and assessed for antigen presentation and processing and for peptide-major histocompatibility complex class I and II (MHCI and MHCII) formation on the cell surface. Interleukin-2 (IL-2) was measured as an indicator of antigen-specific T-cell activation by DCs in coculture. Antigen processing and peptide-MHCII complexes were evaluated by flow cytometry. We observed that ethanol not only suppressed allogeneic peptide presentation to T cells by DCs but also altered presentation of exogenous ovalbumin (OVA) peptide 323-339 to an OVA-specific DO11 T-cell line as well as to OVA-sensitized primary T cells. Smaller amounts of peptide-MHCII complexes were found on the DCs isolated from the spleens of ethanol-fed mice. In contrast to MHCII presentation, cross-presentation of exogenous OVA peptide via MHCI by DCs remained intact. More importantly, ethanol-exposed DCs had reduced B7-DC and enhanced ICOS-L (inhibitory) costimulatory molecule expression. Ethanol inhibits exogenous and allogeneic antigen presentation and affects the formation of peptide-MHCII complexes, as well as altering costimulatory molecule expression on the cell surface. Therefore, DC presentation of peptides in a favorable costimulatory protein environment is required to subsequently activate T cells and appears to be a critical target for the immunosuppressive effects of ethanol.