A convenient, quantitative assay method of branching enzyme (BE) was devised with reduced amylose as the substrate. Using this assay, the properties of the purified branching isoenzymes from maize, BE I, IIa, and IIb, were studied. The method is based on determination of reducing power, by the modified Park-Johnson method, of the chains transferred by BE after they are released from the branched products with isoamylase. The optimum pH of the three enzymes is 7.5, and the optimum temperatures of BE I, IIa, and IIb are 33, 25, and 15-20ยฐC respectively. The specific activities are found to be the highest for BE I and the lowest for BE Ilb, whereas in the conventional assay based on stimulation of unprimed phosphorylase activity, the specific activities are BE IIb > Ha > I. BE I has a lower K, (2.0 PM of the nonreducing terminal) for the reduced amylose of average chain-length (a) 405 than BE IIa (10 PM) and IIb (1 lpM), and the enzyme shows a higher K, for reduced amyloses of smaller aI. Gel-permeation chromatograms on Sephadex G-7SSF of the chains transferred from the reduced amylose indicate that initially the three isoenzymes produce chains of various sizes (dp -8 to > 200), and BE I preferentially transfers longer chains than BE Ila and IIb.