D-25 is a H-2 alloantiserum produced in (B10.D2 X C3H.NB) (H-2d X H-2p)F1 mice after immunization with B10.RIII(H-2r) cells, and which is known to recognize the H-2.25 public antigen on H-2k haplotypes. The "anomalous" reaction of D-25 with a partially purified deoxycholate-solubilized glycoprotein of B10-1 (H-2b) fibrosarcoma was studied with various biochemical techniques. The 125I-labelled precipitates were analysed both in one- and two-dimensional SDS-PAGE and by a partial proteolysis peptide mapping (Cleveland's mapping). The results indicate first that the D-25-related antigen was a genuine H-2 antigen normally associated with the B2-microglobulin, second that this antigen was borne by the Kb but not Db gene products, and finally that D-25 was able to precipitate the same antigen on normal H-2b spleen cells. We conclude that this serum recognized a normal H-2 specificity shared by H-2b and H-2r haplotypes and tentatively identified as the public antigen H-2.54 which for the first time could be assigned to the K but not to the D region of the H-2b haplotype.