We have studied the ligand specificity of a gamma delta T-cell receptor (TCR) derived from a mouse T-cell hybridoma (KN6). KN6 cells reacted with syngeneic (C57BL/6) cells from various origins (splenocytes, thymocytes, peritoneal exudate cells, etc.) and cells from many different mouse strains. KN6 reactivity against cells from a panel of congenic and recombinant mouse strains demonstrated that the ligand recognized by KN6 is controlled by an MHC-linked gene that most probably maps in the TL region. We cloned this gene and formally proved that it does map in the TL region. This gene turned out to be a novel class I gene (designated T22b) belonging to a hitherto unidentified cluster of TL region genes in strain C57BL/6. This gene was expressed in many different tissues and cell types. We also examined the tissue expression of several other TL genes. One of these, the structural gene (T3b) encoding the thymus leukemia (TL) antigen from C57BL/6 mice, was specifically expressed in the epithelium of the small intestine. Since the intestinal epithelium of the mouse is known to be the homing site for a subset of gamma delta T cells (i-IEL) bearing diverse TCR with V7 rearranged gamma chains, we propose that the T3b gene product is part of the ligand recognized by some of the i-IEL. Our data support the idea that gamma delta T cells might be specific for non-classical class I or class I-like molecules and suggest that gamma delta TCR and non-classical MHC co-evolved for the recognition of a conserved set of endogenous or foreign peptides.