The antigen defined by a rat monoclonal antibody directed to a Burkitt lymphoma cell line was identified as globotriaosylceramide [Gal alpha (1 leads to 4)-Gal beta (1 leads to 4)-Glc beta (1 leads to 1)-ceramide]. The antibody demonstrated a strict steric specificity since it did not react with globoisotriaosylceramide [Gal alpha (1 leads to 3)-Gal beta (1 leads to 4)-Glc beta (1 leads to 1)-ceramide], the positional isomer of the antigen associated with the Burkitt lymphoma. Chemical analysis of various Burkitt lymphoma cell lines revealed that the Burkitt lymphoma cells contained more than 100 times as much of the glycolipid antigen as was found in other human lymphoma and leukemia cell lines.