Skin grafts transplanted from B10.HTT donors onto (A.TL × B10)F1 recipients are rapidly rejected despite the fact that the B10.HTT and A.TL strains should be carrying the same H-2 chromosomes and that both the donor and the recipient contain the B10 genome. The rejection is accompanied by a production of cytotoxic antibodies against antigens controlled by the Ir region of the H-2 complex. These unexpected findings are interpreted as evidence for a third histocompatibility locus in the H-2 complex, H-2I, located in the Ir region close to H-2K. The B10.HTT and A.TL strains are postulated to differ at this hypothetical locus, and the difference between the two strains is explained as resulting from a crossing over between the H-2t1 and H-2s chromosomes in the early history of the B10.HTT strain. The H-2 genotypes of the B10.HTT and A.TL strains are assumed to be H-2KsIrs/kSskH-2Dd and H-2KsIrkSskH-2Dd, respectively. Thus, the H-2 chromosomes of the two strains differ only in a portion of the Ir region, including the H-2I locus. The B10.HTT(H-2tt) and B10.S(7R)(H-2th) strains differ in a relatively minor histocompatibility locus, possibly residing in the Tla region outside of the H-2 complex. © 1974 Springer-Verlag New York Inc.