We have used radiochemical techniques to characterize the heavy chain (Mr 46,000) of the murine H-2Kk histocompatibility antigen in terms of six fragments (I-VI) obtained after cleavage of the polypeptide chain with CNBr. The tentative order of the fragments, which account for more than 90% of the heavy chain, was assigned by radiochemical sequence analysis of the intact heavy chain and of each purified CNBr fragment and by analysis of the CNBr fragments obtained from the large papain fragment of the heavy chain. Treatment of cells with tunicamycin yielded H-2 molecules with heavy chains of molecular weight 40,000, suggesting that the carbohydrate moieties have a combined molecular weight of approximately 6000. CNBr cleavage of H-2Kk heavy chains labeled with [3H]fucose indicated that the carbohydrate moieties are located on fragments II and IV.. Incubation of cells with 32PO4 gave H-2 molecules with radioactive phosphoserine in the carboxyl-terminal CNBr fragment (VI) of the heavy chain and in the fraction containing beta 2-microglobulin. Sequence analysis of each CNBr fragment intrinsically labeled with 3H- and 35S-labeled amino acids identified a total of 87 residues in the H-2Kk heavy chain. The sequence closely resembles that of the H-2Kb molecule, and the 11 differences are scattered throughout the polypeptide chain. Comparison with HLA sequences indicates that the two allelic H-2 sequences are more closely related to each other (88% identity) than either is to the HLA-B7 or A2 antigens (approximately 70%). Similarly, the nonallelic HLA antigens are more closely related to each other (83%) than either is to the H-2Kk or H-2Kb molecules.