A 290-kilobase-pair chromosomal segment containing the genes encoding the human class 1 major histocompatibility complex molecules HLA-B and HLA-C as well as a class 1 pseudogene has been isolated on three overlapping yeast artificial chromosome (YAC) clones. One YAC clone contains both the HLA-B and HLA-C genes. These loci are located ≈85 kilobase pairs apart, each in close association with a CpG island. Southern blotting and nucleotide sequencing showed no evidence of alteration of the structure of the cloned DNA in the YACs. End fragments from the YAC inserts have been isolated and used to confirm the overlaps between clones. These fragments can also serve as polymorphic markers for structural analysis of the major histocompatibility complex. Our data show that YAC cloning offers an attractive alternative for analysis of the structures of large gene complexes such as HLA.