The HLA-DR antigen expressed on the surface of the human melanoma cell line SK-MEL-37 was characterized and compared with the HLA-DR antigen from the MU B lymphoblastoid cell line originating from the same individual. The HLA-DR heavy chain from SK-MEL-37 cells had an apparent mobility on SDS-PAGE slightly slower than that isolated from MU cells. In contrast, the HLA-DR light chains and the HLA-A,-B heavy chains from the two cell lines had identical mobilities. Double-labeled tryptic peptide mapping and limited N-terminal sequencing showed that the SK-MEL-37 HLA-DR antigen, like all previously examined B lymphoblastoid cell HLA-DR antigens, was homologous to the murine I-E/C subregion antigens and that the mobility difference of the SK-MEL-37 HLA-DR heavy chain was not attributable to differences in the primary structure of the polypeptide. Treatment of the cells with tunicamycin abolished the m.w. difference, suggesting that it was due to a change in glycosylation in SK-MEL-37. This was confirmed by analysis of the glycopeptides from pronase-digested HLA-DR light and heavy chains and HLA-A,B heavy chains purified from the two cell types. The results suggest 1) there is a difference in asparagine-linked oligosaccharide processing in the two cell types, with more of the larger complex glycans synthesized in the melanoma cells than in the B lymphoblastoid cells, 2) the effect is more pronounced with HLA-DR heavy chains than with HLA-DR light chains or HLA-A,B heavy chains, and 3) the oligosaccharide size difference is not solely due to sialic acid content.