T cells specific for foreign antigen recognize a complex of peptides and self-major histocompatibility complex (MHC) molecules and can also cross-react with allo-MHC molecules. It remains controversial, however, what alloreactive T cells exactly recognize. It has been proposed that alloreactive T cells recognize endogenous peptides presented by allo-MHC molecules. To test this hypothesis, we examined an influenza virus-specific T cell clone (6H5), specific for neuraminidase N2 and restricted by HLA-DR1. In the absence of influenza virus, this clone cross-reacted with HLA-DR1Dw1+ but not with HLA-DR1Dw20+ Epstein-Barr virus-transformed lymphoblastoid cells (B-LCL). Cold target inhibition experiments and the rearrangement pattern of the T cell receptor beta chain indicated that 6H5 was a monoclonal T cell population most likely using the same T cell receptor for both responses. To determine whether determinants other than HLA-DR1Dw1+ B-LCL or activated B cells, but, surprisingly, not to other cell types expressed HLA-DR1Dw1, including monocytes and transfected L cells. These experiments further support the concept that recognition of allogeneic MHC (in this case HLA-DR1Dw1) may result from a cross-reactivity of T cells specific for a complex of foreign antigen and self-MHC (neuraminidase N2 and HLA-DR1Dw20). Furthermore, allorecognition of T cell clone 6H5 appears to depend upon the recognition of a complex of allogeneic MHC and a cell-type specific endogenous peptide presented by activated B cells.