Polypeptide phosphorylation and sialylation of the glycan moieties contribute to the charge heterogeneity of the class I major histocompatibility complex glycoproteins. The present study demonstrates that a unique acidic modification unrelated to phosphorylation or glycosylation also affects the charge heterogeneity of the H2-Kk heavy chain of BW5147 lymphoma cells. In vitro cultivation of BW5147 cells results in changes in charge heterogeneity of the H2-Kk heavy chains due to the unique acidic modification. Sequential papain digestion of the 45 000 Mr H2-Kk glycoprotein yields a 42 500 Mr glycopolypeptide initially, followed by production of a 39 000 Mr glycopolypeptide. Results from experiments designed to localize and characterize the novel acidic modification suggest that the modification resides in the segment of the H2-Kk polypeptide located between the two papain cleavage sites. This portion of the polypeptide consists of the transmembrane region and part of the cytoplasmic domain of the H2-Kk heavy chain. At steady state, 25% of the total cell surface H2-Kk possesses this modification. In addition, the modification is mutually exclusive with the phosphorylation of the H2-Kk heavy chain at Ser-333. The possible biological significance of the novel modification of class I antigens is discussed.