The two DR1-associated cellular specificities Dw1 and Dw20, as well as DR'Br' (Dw'BON'), cannot be unequivocally assigned by serological typing or restriction fragment length polymorphism (RFLP) analysis. We have developed and compared two polymerase chain reaction-based (PCR) typing methods for distinguishing these DRB1 alleles; allele-specific amplification of DRB1*01 alleles followed by an agarose gel electrophoresis detection step and group-specific DRB1*01 amplification followed by hybridization with sequence-specific oligonucleotide probes. The two typing strategies gave completely concordant results in the 33 DRB1*01-positive and the 46 DRB1*01-negative individuals and cell lines studied. No false-negative or false-positive typing results were obtained. All possible heterozygous combinations of the DRB1*0101-0103 alleles could be distinguished by both typing methods. DRB1*01 subtyping by allele-specific PCR amplification was performed in less than 3 hours, including PCR amplification, detection and interpretation steps. The technique will be a valuable complement to DR typing by serology and RFLP analysis. Allele-specific DRB1 amplifications or group-specific amplifications followed by directed allele-specific amplifications of DRB1 alleles, typing based on the absence or presence of amplified products, may well prove to be the technical innovation that will firmly establish PCR-based DR typing in routine clinical tissue typing.