A genomic clone specifying a new HLA class II antigen beta chain, DQB3, was isolated from a human genomic phage library using a DQB1 cDNA probe under low stringency conditions. Southern hybridization and nucleotide sequence analyses identified the beta 2 domain exon (exon 3) with several deleterious mutations and the CP-TM-CY exon [connecting peptide, transmembrane, and cytoplasmic regions, (exon 4)], but the first, second, and fifth exons encoding the 5' UT-leader, the beta 1 domain, and the 3' UT domain of normal beta chains, respectively, were entirely missing. The nucleotide sequences of these two exons were distinct from those of other class II beta chain genes, but slightly more related to the DQB1 and DQB2 genes than to other class II genes. The DQB3 sequence mapped between DQA2 and DQB1, 15 kb upstream from DQA2, by analysis of overlapping cosmid clones. This mapping was supported by the fact that Taq I, Msp I, and Bam HI DQB3 polymorphisms were perfectly correlated with the DQA2 polymorphism and not with any polymorphisms in the DR or DQ subregion, suggesting the presence of a hot spot for recombination between DQB3 and DQB1.