Single-strand conformational polymorphism (SSCP) gel electrophoresis and DNA sequencing were used to characterize the second exon of the horse DRB homologue as well as to identify eight new DRB alleles. The SSCP gels presented a complex pattern, with phenotypes exhibiting between 4 and 13 bands. The DRB SSCP patterns were studied for two families (6 to 13 bands per pattern). For both families, the patterns showed simple Mendelian inheritance. The polymerase chain reaction products from two individuals possessing homozygous major histocompatibility complex (MHC) alleles by descent were cloned and retested on SSCP gels. All bands derived from the genomic DNA amplification could be accounted for with bands derived from the cloned DNA amplification products. The results were consistent with three DRB loci, though this number may be variable within the domestic horse population. Gene sequences were variable among the different products, and we were unable to assign locus designations for particular sequences. Amplification of cDNA library material derived from one of the individuals who is MHC homozygous by descent showed an SSCP profile suggesting that all three DRB loci are transcribed into mRNA.