The murine monoclonal antibody (MAb) BW494 defines a carbohydrate epitope on a mucin-type glycoprotein which is expressed on the majority of well-differentiated adenocarcinomas of the pancreas. In this contribution we present evidence for the detailed structural requirements of the BW494 interaction with the glycan portions on (neo)glycoproteins or (neo)glycolipids as revealed by solid-phase binding assays and inhibition assays of BW494 binding to synthetic antigens or to the affinity-isolated cancer mucin CA494. The observed cross-reactivity of spacer-linked Gal beta 1-3GalNAc beta (TF-beta) and Gal beta 1-3GlcNAc beta (type 1 chain) is indicative of an epitope that comprises a terminal Gal beta 1-3HexNAc unit. The active conformation of this epitope is critically influenced by the chemical environment of the terminal disaccharide, i.e. by adjacent aglycon or spacer moieties or by substitution with further sugar residues at the reducing end. Although a strong enhancement of antibody binding is observed for the type 1 chain derived Lea antigen, MAb BW494 is distinct in its reactivity from Lea-specific antibodies.