Abstract: We have identified locus-specific sequences in the first and third introns flanking the polymorphic second and third exons of HLA class I genes. PCR primers derived from these conserved sequences produced DNA fragments of the expected sizes for each of the HLA-A, -B, and -C loci in the amplification of genomic DNA. PCR products generated using each of the locus-specific sets of primers displayed exquisite locus specificity, as assessed by hybridization with oligonucleotide probes specific for ten classical and non-classical HLA class I genes. Amplification with these primer sets was effective and specific for the HLA alleles tested under the given PCR conditions. When hybridized with oligonucleotides derived from shared polymorphic sequence motifs, reaction patterns of PCR products from each locus were precisely as expected from published or database sequences. Chemiluminescent signals generated from digoxygenin-ddUTP-labeled probes were even for all samples and as strong as those obtained in MHC class II typing. These locus-specific primer sets derived from intron sequences provide an effective means to amplify genomic DNA which will facilitate PCR-based HLA class I typing methods. This will also allow HLA class I typing to be conducted with greater precision, at lower cost, and faster than previously described class I typing methodologies.