17 HLA class I genes have been isolated from the genome of B-lymphoblastoid cell line 721. Sequence analysis and transfection studies indicate that three genes, in addition to those encoding the HLA-A, -B, and -C antigens can direct the synthesis of a class I α protein. Using gene-specific DNA probes to analyze the presence of restriction fragment-length polymorphisms within a large pedigree and in panel of HLA deletion mutant cell lines, we show that two of these genes, designated HLA-G and HLA-F, are lcoated on the short arm of chromosome 6 telomeric to the HLA-A locus. The third expressed non-A, -B, and -C class I gene, HLA-E, is located between HLA-A and HLA-C. In addition, the remaining 11 class I pseudogenes and gene fragments are localized relative to established markers on chromosome 6p.