Cloning and characterization of classical MHC class I coding sequences of the laboratory rat Rattus norvegicus has been reported so far for only four haplotypes, RT1a, RT1(1), RT1n, and RT1u. In all four cases, only one RT1.A classical class I molecule was found. Here we report that, in contrast, the RT1c haplotype expresses two different classical class I molecules. Using recombinant rat strains, we find that allotypic serologic determinants carried by the two molecules map to the RT1.A region, and so we have named them RT1.A1c and RT1.A2c. Multiple clones of functional cDNAs for each of these two molecules were isolated using a recently developed PCR-based expression-cloning method. Using a panel of 20 RT1.Ac-reactive mAb, we find that six recognize RT1.A1c, seven recognize RT1.A2c, and seven recognize both. We also show that both molecules are recognized and distinguished by primary alloreactive cytotoxic T lymphocytes, and that they correspond to identifiable and distinct molecular species in cells that express RT1c naturally. These data all concur to demonstrate that the RT1.Ac region carries two different loci, each of which encodes a functional classical class I molecule.