Murine anti-Ia sera were tested for their reactivity with rat peripheral lymphocytes. Extensive cross-reactions were observed in a complement-dependent cytotoxicity assay. In the majority of strain combinations, these reactions were specific for B cells. We demonstrated by absorption analyses that (1) the reaction was specific, (2) B cells from all rat strains reacted with anti-I-Ek serum, and (3) the anti-I-A sera defined five new specificities. These antisera can also immunoprecipitate class II (Ia-like) molecules from radiolabeled, detergent-solubilized cell extracts. The rat equivalents of molecules encoded in the I-A and I-E subregions in mice can be isolated from all rat strains studied. The alpha and beta subunits of the rat class II molecules were polymorphic when analyzed by sodium dodecyl sulfate (SDS) polyacrylamide electrophoresis. This observation allows the characterization of rat alloantisera or monoclonals of unknown specificity by comparing them to the defined reactivity of the mouse anti-Ia sera.