Monoclonal antibodies specific for the rat major histocompatibility complex (MHC) class I antigens RT1.An, RT1.Au, and RT1.Eu were used for immunoprecipitation of antigens biosynthetically radiolabeled with 14C- or 3H-labeled arginine, lysine, and tyrosine; with arginine or tyrosine alone; and with or without tunicamycin in the culture medium. Heavy chains of the glycosylated and unglycosylated antigens were purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their tryptic and chymotryptic peptides were compared by high performance liquid chromatography. The antigens coded by the same locus in two different haplotypes (An and Au) differed by 30%, whereas the products of two different loci in the same haplotype (Au and Eu) differed only by 1-3%. Comparative analysis of the data for samples labeled with single amino acids indicated that two amino acids in Au have been substituted by an arginine and probably by a tyrosine residue, respectively, in Eu. The high degree of homology between the products of the A and E loci in the same haplotype accounts for the difficulty in detecting recombinational events within the MHC of the rat by classical serological approaches.