This work was designed to investigate the structural features required for a branched xyloglucan nonasaccharide (XG9; composition: glucose(4)xylose(3)galactose(1)fucose(1)) to exhibit anti-auxin activity in the pea (Pisum sativum L.) stem segment straight-growth bioassay. Oligosaccharides were prepared by cellulase-catalyzed hydrolysis of Rosa xyloglucan, and tested for auxin antagonism. The quantitatively major hepta-, octa-, and decasaccharides (XG7, XG8, and XG10) showed no antiauxin activity at the concentrations tested and did not interfere with the antiauxin effect of 10(-9) molar XG9 when coincubated at equimolar concentrations. The results indicate that the XG9-recognition system in pea stem segments is highly discriminating. A terminal alpha-l-fucose residue is essential for the antiauxin activity of XG9 and a neighboring terminal beta-d-galactose residue can abolish the activity; possible reasons for the effect of the galactose residue are discussed. A sample of XG9 extensively purified by gel-permeation chromatography followed by paper chromatography in two solvent systems still exhibited antiauxin activity with a concentration optimum around 10(-9) molar. This diminishes the likelihood that the antiauxin activity reported for previous nonsaccharide preparations was due to a compound other than XG9.