MHC class II-positive late endosomes of enterocytes are thought to be involved in antigen presentation to CD4(+) T cells. In contrast to enterocytes of BALB/c mice, severe combined immunodeficiency (SCID) enterocytes lack MHC class II expression and fail to transport internalized ovalbumin (OVA) into late endosomes. IFN-gamma is known to induce MHC class II in enterocytes and antigen targeting to late endosomes in macrophages. In this study, we investigated the influence of IFN-gamma and MHC class II on the processes of antigen traffic in enterocytes. Subcellular targeting of OVA and MHC class II expression within enterocytes were examined in SCID, IFN-gamma-treated SCID, BALB/c and C57BL/6 MHC class II knockout (KO) mice after a single feed with OVA. Sorting of OVA into late endosomes was found in enterocytes from BALB/c, C57BL/6 KO and IFN-gamma-stimulated SCID mice, but not from untreated SCID mice. MHC class II expression was restricted to enterocytes of IFN-gamma-treated SCID and BALB/c mice, present at basolateral membranes and within endosomal compartments. These enterocytes further revealed colocalization of class II antigens and OVA in endosomes. We suggest that antigen trafficking into late endosomes of enterocytes is mediated by IFN-gamma and occurs in the absence of MHC class II.