When human class I cDNA clones containing coding sequences are used to probe genomic DNA, 15-20 fragments, each containing a complete class I gene or pseudogene, are seen. Identification of which genomic DNA segments encode the HLA-A and -B antigens has to date required transfection of mouse L cells with cloned class I genes or analysis of HLA loss mutants. In this report we show that under high-stringency conditions, probes constructed from the 3'-untranslated region can be used to specifically identify the segments of DNA that encode the HLA-A and -B antigens in the human lymphoblastoid cell line 721. Examination of DNA from unrelated individuals indicates that these probes are locus specific and will permit identification of HLA-A and -B genes in the population.