RT1.B Class II molecules, comparable to I-A and I-E molecules in mice, have been characterized by two-dimensional (2D) gel electrophoresis for seven rat strains expressing different RT1 haplotypes. RT1.B molecules were immunoprecipitated from radiolabeled rat lymphocyte preparations with mouse monoclonal antibodies reactive with mouse I-Ab antigens and I-Ek antigens. For each RT1 haplotype, two I-A-like subunits, alpha and beta, and two I-E-like subunits, gamma and delta, have been identified. 2D gel analysis of RT1.B molecules from different strains in paired combinations reveals that, with a few notable exceptions, the alpha, beta, and delta subunits associated with different RT1 haplotypes are characterized by unique 2D gel spot patterns. In contrast, the gamma subunits of all RT1.B haplotypes appear identical. This evidence confirms and extends our previous analysis of RT1.B molecules by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and suggests a high level of polymorphism for genes in the RT1.B region, with the exception of the RT1.Bc gene encoding gamma. Interestingly, three rat strains appear exceptional in the identity of their alpha, gamma, and delta subunits, which suggests that these strains express closely related RT1 haplotypes derived from a common progenitor.