The major histocompatibility complex of the rat (RT1) has been poorly characterized with respect to the number, linkage, and polymorphism of class I genes. To estimate the number of class I RT1 genes and the relative extent of their polymorphism, we performed Southern blot analysis with liver DNA from rat strains expressing eight RT1 haplotypes. After digestion with EcoRI and BamHI, the DNA was separated on agarose gels, blotted onto nitrocellulose, and hybridized with mouse H-2 cDNA probes, pH-2III and pH-2IIa. Ten to 20 EcoRI and 13 to 20 BamHI bands hybridized with pH-2III and pH-2IIa; restriction fragment length patterns were observed to be highly polymorphic. The restriction fragments associated with different RT1 haplotypes differed by 17-70%; this range is similar to the differences observed between mouse H-2 haplotypes. The same restriction fragment pattern was observed in DNA from three different rat strains sharing the same RT1 allele, confirming that the patterns were RT1-associated. Further, the RT1l and RT1lvl haplotypes, which differ at a single previously identified RT1-linked locus, were associated with EcoRI restriction pattern differences of 39-50%, confirming the supposition that RT1 class I genes identified by previous serological and T-cell-mediated assays have identified only a minority of the actual number of RT1-linked class I genes. In summary, the results reported in this communication demonstrate that the RT1 complex encompasses a large family of highly polymorphic class I genes similar to the H-2 and HL-A complexes of mouse and man.