Ia antigens from two inbred strains of rats have been characterized by two-dimensional gel (IEF/SDS PAGE) and microsequence analysis. Rat Ia antigens are isolated from biosynthetically radiolabeled spleen cells using semicongenic alloantisera. Rat Ia antigens are composed of two components on SDS-PAGE as are their mouse, human, and guinea pig counterparts. The larger polypeptide (α) has an apparent molecular weight of 33,000. The α polypeptides of AgB3 and AgB4 haplotypes show striking homology in their partial N-terminal amino acid sequences to the mouse ECαk and ECαd polypeptides and to human P34. The rat B3 and B4 α polypeptides are identical at six of eight positions which can be compared. The smaller polypeptide (β) has an apparent molecular weight of 28,000. Preliminary data on the β polypeptides suggest the existence of multiple molecular species for each haplotype. Sequences resembling mouse IA-β and I-EC-β polypeptides may be present.