The major histocompatibility complex (MHC) of the rat (RT1 complex) encodes two sets of class II molecules referred to as RT1.B and RT1.D. The RT1.B alpha gene was isolated for a Sprague-Dawley (RT1b) rat genomic library using a rat RT1.B alpha chain cDNA as a hybridization probe. The coding and the majority of the intron DNA sequence was determined. The structure of the RT1.B alpha gene is equivalent to that of H-2 and HLA alpha chain genes. Comparison of the nucleotide and predicted amino acid sequences of the RT1.B alpha gene with those of the H-2 and HLA genes revealed a high degree of overall sequence conservation. However, two regions of the first external domain (alpha 1), residues 19-23 and 45-78, exhibit marked sequence variation. Two blocks of conserved nucleotide sequence were identified in the 5' promoter region of the RT1.B alpha gene that have been described in all MHC class II genes sequenced to date. These conserved sequences may be involved in the coordinate regulation of expression of class II genes. The cloned RT1.B alpha gene was efficiently transcribed when transfected to mouse L cells.