The major histocompatibility complex of the rat (RT1) contains genes that code for two class II histocompatibility antigens. The r12 rat strain (WRC) was derived from a mating which revealed a genetic recombination that defines the two loci, RT1.B and RT1.D, that code for the class II antigens. The RT1.B and RT1.D antigens of the RT1 complex are protein dimers consisting of an alpha and beta glycoprotein chain and are homologous to I-A and I-E genes, respectively, in the H-2 complex of the mouse. We have performed Southern blot analysis on liver DNA from the r12 and parental strains to examine the precise location of the recombinant event. After digestion with restriction enzymes, the DNA was separated on agarose gels, blotted onto nitrocellulose, and hybridized with mouse H-2 cDNA probes specific for I-A alpha and beta genes. The pattern of restriction fragment polymorphisms demonstrated that the site of recombination is between the RT1.B alpha and the RT1.B beta genes. As a result of these findings, we suggest that the sequence of genes within the RT1 complex consists of RT1.A ... RT1.B beta ... RT1.B alpha ... RT1.D (alpha, beta) ... Other class I genes, possibly corresponding to mouse Qa and Tla-like genes, were also apparent in these experiments.