H-2d-encoded gene products were analyzed as restriction antigens for anti-vesicular stomatitis virus (VSV) cytotoxic T lymphocytes (CTL). Cold target competition experiments revealed that VSV recognition was H-2D region-restricted; H-2K-end-restricted recognition of VSV could not be demonstrated. That VSV is not recognized in the context of K-region-encoded gene products is also supported by the observation that H-2dm1 and H-2dm2 mice, strains that contain H-2Kd but have an alteration in H-2L and/or H-2D/L, are nonresponders in the CTL assay. Two different lines of evidence eliminated H-2Dd, H-2Md, and H-2Rd as the restriction antigens: (a) H-2dm2-VSV inhibitors that express H-2Dd and H-2Md did not block the lysis of P815-VSV targets by Balb/c anti-VSV killer cells, and (b) a hybridoma specific for H-2Dd failed to inhibit killer cell activity in this same effector/target combination. However, two monoclonal antibodies specific for H-2Ld but not H-2Rd completely blocked anti-VSV cytotoxic activity. Taken together, in the H-2d haplotype, anti-VSV CTL recognize VSV solely in the context of the H-2Ld molecule. This is the first demonstration of the exclusive use by a mouse stain of the H-2L molecule only for H-2-restricted recognition, and thus supports the notion that H-2L plays a major role in restricting antigen specific recognition. Finally, the fact that an anti-H-2Ld monoclone completely blocked an H-2dm2 anti-BALB/c CTL response indicates that H-2R, a molecule absent in H-2dm2 anti-BALB/c CTL response indicates that H-2R, a molecule absent in H-2dm2 but not BALB/c, does not sensitize H-2 alloreactive CTL.