Two monoclonal antibodies directed to the Type II carbohydrate chain of glycosphingolipids have been prepared by the murine hybridoma technique. Their reactivity was determined by liposome lysis, plate-binding assay, complement fixation, and hemagglutination inhibition. One antibody was specific for glycolipids having the nonreducing terminal N-acetyllactosamine (Gal beta 1 leads to 4GlcNAc beta 1 leads to R) structure and did not react with glycolipids having either a Type I chain (Gal beta 1 leads to 3GlcNAc beta 1 leads to R), a ganglio-series structure (Gal beta 1 leads to 3GalNAc beta 1 leads to R), or a sialosyl or fucosyl substitution at the N-acetyllactosamine residue. This antibody readily agglutinated adult human erythrocytes of all ABO types but did not agglutinate umbilical cord cells. The other antibody reacted with the Type II chain H structure (Fuc alpha 1 leads to 2Gal beta 1 leads to 4GlcNAc beta 1 leads to R) but reacted only weakly with the Type I chain H (Fuc alpha 1 leads to 2Gal beta 1 leads to 3GlcNAc beta 1 leads to R) and ganglio-series H (Fuc alpha 1 leads to 2Gal beta 1 leads to 3GalNAc beta 1 leads to R) structures. At 37 degrees C this antibody agglutinated human erythrocytes of types O and A2 but was unable to cause detectable agglutination of types B and A1. Because of their narrow, well defined specificity, these monoclonal anticarbohydrate reagents will be useful in the study of the distribution, quantity, and function of specific carbohydrates on the cell surface.