The major histocompatibility complex of the rat (RT1) encodes the class II molecules involved with antigen presentation and cell to cell communication. The organization of these class II genes has been studied by Southern blot hybridization using genomic DNA from inbred and recombinant rat strains digested with various restriction endonuclease and hybridized under stringent conditions with probes for mouse class II and human class II genes. Analysis of the restriction fragment length polymorphisms has mapped the class II genes relative to each other. We have confirmed the order of the alpha- and beta-chain genes in the RT1.B region, mapped the RT1.D region relative to RT1.B and showed that it has alpha- and beta-chain loci, and identified a new HLA-DP-like locus, RT1.H, to the RT1.A side of RT1.B. The RT1.H alpha and RT1.H beta genes map to the region around the recombination point in R22, and there appears to be a hot spot of recombination in RT1.H. The H beta and D beta genes have high levels of polymorphism; B beta, B alpha, and H alpha have intermediate levels of polymorphism, and D alpha has a low level of polymorphism.