Monoclonal antibody D1.1 originally prepared against the B49 cell line derived from a rat brain tumor was shown to react with a ganglioside present in fetal rat brain. We have found that this antigen is also present in human malignant melanoma tumors as well as many melanoma cell lines. The ganglioside from human melanoma cell lines migrates between GM1 and GM2 on one-dimensional thin layer chromatography. Analysis by two-dimensional thin layer chromatography with intermediate ammonia treatment suggests that the ganglioside contains one or more base-labile O-acyl esters. Mild base hydrolysis under conditions known to remove O-acyl esters results in complete loss of antigenic reactivity. Thus, the alkali-labile moiety is a critical component of the epitope recognized by the antibody. Analysis of the sialic acids of total gangliosides from [6-3H]glucosamine-labeled melanoma cells showed that approximately 10% of these molecules are O-acylated. Similar analysis of the purified ganglioside showed that greater than 30% of the sialic acids comigrated with authentic 9-O-acetyl-N-acetylneuraminic acid. The antibody did not cross-react with normal human skin melanocytes nor with any of a large number of normal human adult and fetal tissues. The antibody also did not react with numerous other malignant cell lines studied. These findings suggest that the antigenic epitope defined by antibody D1.1 contains an O-acylated sialic acid and may arise from aberrant O-acetylation occurring in human malignant melanoma cells.