The human fibroblast cell line, M1, expressing the products of transfected DRA and DRB1*0101 genes (M1-DR1) was unable to present intact influenza antigens to a series of DR1-restricted human T cell lines and clones, but was fully able to present synthetic peptides for T cell recognition. In contrast, M1-DR1 cells infected with live influenza virus were recognized by two polyclonal hemagglutinin- or whole virus-specific T cell lines and one of four T cell clones. This difference could not be accounted for simply by the ability of infectious virus to overcome a defect in antigen uptake by the M1-DR1 cells, in that direct studies of endocytosis showed that the M1 cells were more efficient than human B cells in the internalization of exogenous protein. These data suggested that the M1 cells were unable to present exogenous antigens but were capable of loading major histocompatibility complex (MHC) class II molecules with peptides derived from endogenous antigens. To investigate this further, the M1-DR1 cells were super-transfected with a cDNA encoding the p33 and p35 forms of the human invariant chain (Ii). Expression of the Ii chain was detected by intracytoplasmic staining of transfectants, and by metabolic labeling. Equimolar amounts of the p33 and p35 forms were detected, and the high level of p35 Ii was reflected by extensive retention of Ii protein in the endoplasmic reticulum. Addition of the Ii chain led to no recovery of presentation of intact antigens with DR1, but inhibited the presentation of live virus. These data indicate that MHC class II molecules in the M1-DR1 cells can be loaded with peptides derived from endogenous proteins, possibly in the biosynthetic pathway, and that the Ii chain has a role in limiting this route of class II antigen presentation.