Three forms of lectin (components I, II, and III) from lima beans (Phaseolus lunatus) have been purified on an affinity support containing the synthetic type A blood group trisaccharide alpha-D-GalNAc-(1 leads to 3)-[alpha-L-Fuc-(1 leads to 2)]-beta-D-Gal-(1 leads to). Conversion of components I and II to component III has been achieved by reduction in 10(-2) M dithiothreitol. Isoelectric focusing of lima bean lectin in the presence of 8 M urea and beta-mercaptoethanol revealed charge heterogeneity of the lectin subunits. Three major subunit classes of apparent pI 7.05, 6.65, and 6.45, designated alpha, beta, and alpha', respectively, were identified; they occur in a relative abundance of 2:5:3. Green lima beans harvested before maturity lacked the alpha' subunit (pI 6.45) which appears to accumulate during seed maturation. The three subunits are glycoproteins of identical size and immunochemical reactivity. Identical NH2-terminal sequences were found for the three subunits. Amino acid analysis and tryptic peptide mapping indicated that the observed charge heterogeneity is probably due to differences in the primary structure of the subunits. Studies of subunit composition of charge isolectins provided evidence of nonrandom subunit assembly. A model is proposed involving pairing of a pI 6.65 subunit with either a pI 7.06 or 6.45 subunit to form dimeric units. Possible roles for subunit heterogeneity and ordered subunit assembly in determining the metal and sugar binding properties of lima bean lectin are discussed.