The amphibian Xenopus laevis is the most primitive vertebrate in which the major histocompatibility complex (MHC) has been defined at the biochemical, functional, and molecular genetic levels. We previously described the isolation and characterization of cDNA clones encoding X. laevis MHC class II beta chains. In the present study, genomic clones encoding class II beta chains were isolated from X. laevis homozygous for the MHC f haplotype. Three class II beta chain genes, designated Xela-DAB, Xela-DBB, and Xela-DCB, were identified. Sequence analysis of these genes showed that Xela-DBB and Xela-DCB correspond to the previously characterized cDNA clones F3 and F8, respectively, whereas Xela-DAB encodes a third, hitherto unidentified class II beta chain of the MHC f haplotype. As a representative of X. laevis class II beta chain genes, the Xela-DAB gene underwent detailed structural analysis. In addition, the nucleotide sequence of Xela-DABf cDNA clones was determined. The Xela-DAB gene is made up of at least six exons, with an exon-intron organization similar to that of a typical mammalian class II beta chain gene. The 5'-flanking region of the Xela-DAB gene contains transcriptional control elements known as X1, X2, and Y, but lacks typical TATA or CCAAT boxes. A notable feature of the X. laevis class II beta chain genes is that the sizes of the introns are larger than those of their mammalian counterparts. As assessed by northern blot analysis, the three class II beta chain genes had similar expression patterns, with the highest level of transcription detected in the intestine. Identification of the Xela-DAB, -DBB, and -DCB genes is consistent with our previous observations, which suggested that the MHC of the tetraploid frog X. laevis is diploidized at the genomic level and contains three class II beta chain genes per haplotype that cross-hybridize to one another under reduced stringency conditions.