The purpose of this study was to isolate mouse monoclonal antibodies (MAbs) recognizing Gal alpha 1-4Gal beta 1-4Glc (Pk, CD77), which has been described as a Burkitt's-lymphoma-associated antigen. Three IgGI MAbs reactive with Pk were developed using a synthetic glycoconjugate as immunogen and a filter immunoplaque screening assay. One MAb (PK67) was characterized by immuno-thin-layer chromatography, ELISA and competition assays using neutral glycolipids from various sources and a variety of carbohydrates and glycoproteins. Epitope analysis showed that all 3 carbohydrates moieties are required in PK67 binding. No cross-reactivity was observed with closely related carbohydrate structures, with the exception of Gal alpha 1-4Gal beta 1-4GlcNAc. However, the reactivity with this structure was several orders of magnitude lower than with Pk. Immunohistochemical analysis of fresh-frozen tissue specimens showed that Gb3 is widely expressed: prominent staining was observed in many normal tissues, e.g., kidney and gastric tissue and on capillary endothelial cells. In lymph nodes, very weak staining of a few B cells was observed. Flow cytometric analysis of hematopoietic neoplasms showed Gb3 expression on B-cell neoplasms, particularly those with committed B-cell phenotype indicating that, in addition to earlier reports of Gb3 expression on Burkitt's lymphoma, Gb3 is present on other committed B-cell neoplasms.