A lectin (carbohydrate-binding protein) has been found in extracts of a number of axenically grown trophozoites of Entamoeba histolytica strains. The strains grown in TYI-S-33 medium (Diamond et al., Trans. R. Soc. Trop. Med. Hyg. 72: 431-432, 1978) were HK-9, 200:NIH, and HM-1:IMSS. Strain HU-1:MUSC (HSC) was grown monoxenically in the same medium. The amoebic lectin agglutinated glutaraldehyde-fixed erythrocytes. This activity was pH dependent and heat and oxidation sensitive, and was destroyed by proteolysis upon autoincubation. The relative agglutinating potency of the different strains of amoebae was investigated. Strain HSC had the highest specific activity (210 U/mg of protein), and strain HM-1 had the lowest (14 U/mg). One unit of hemagglutinating activity is defined as the amount of lectin present in 1 ml of extract which will agglutinate 1 ml of 4% erythrocytes. Upon subcellular fractionation of the lectin present in extracts of strain HK-9, two-thirds of the activity was detected in the soluble, nonsedimentable (100,000 x g, 60 min) fraction. Partial hydrolysate of chitin was found to inhibit the hemagglutinating activity. Among the oligosaccharides of N-acetylglucosamine, the trimer and tetramer were the most potent inhibitors. The lectin was purified approximately 300-fold by a one-step affinity chromatography on a chitin column. The loading and elution from the column were based on the pH dependence of the lectin activity.