We previously reported a simple and rapid method for HLA-DQA genotyping by digestion of polymerase chain reaction-amplified DQA genes with allele-specific restriction endonucleases. Here we report the application of this method to DP genotyping. The second exon of the HLA-DPB genes was selectively amplified from genomic DNAs of 72 HLA-D homozygous B-cell lines by the polymerase chain reaction method. Amplified DNAs were digested with ApaI, SacI, BstUI, FokI, and RsaI, which can recognize allelic sequence variations in the polymorphic segments of the DPB second exon and then subjected to electrophoresis in polyacrylamide gels. Sixteen different polymorphic patterns of the restriction fragments were found, and twelve were identical to patterns predicted from the known DNA sequences correlating with each HLA-DPw specificity defined by cellular typing. The other four patterns were distinct from those of the known DPw specificities, suggesting the presence of novel DP alleles. This polymerase chain reaction-restriction fragment length polymorphism method provides a simple and rapid technique for accurate definition of HLA-DP types at the nucleotide level, replacing the technically demanding method of primed lymphocyte typing.