An alpha-fucosidase that releases fucosyl residues from oligosaccharide fragments of xyloglucan, a plant cell wall hemicellulosic polysaccharide, was purified to homogeneity from pea (Pisum sativum) epicotyls using a combination of cation exchange chromatography and isoelectric focusing. The alpha-fucosidase has a molecular mass of 20 kDa according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The alpha-fucosidase has an isoelectric point of 5.5. The substrate specificity of the alpha-fucosidase was determined by high performance anion exchange chromatographic analysis of oligosaccharide substrates and products. The enzyme hydrolyzes the terminal alpha-1,2-fucosidic linkage of oligosaccharides and does not cleave p-nitrophenyl-alpha-L-fucoside. The enzyme does not release measurable amounts of fucosyl residues from large polysaccharides. The subcellular localization of alpha-fucosidase in pea stems and leaves has been studied by immunogold cytochemistry. The alpha-fucosidase accumulates in primary cell walls and is not detectable in the middle lamella or in the cytoplasm of 8-day-old stem tissue and 14-day-old leaf tissue. alpha-Fucosidase activity was readily detected in extracts of 8-day-old stem tissue. No significant alpha-fucosidase activity or immunogold labeling of the alpha-fucosidase was detected in 2- and 4-day-old stem tissue indicating that production of alpha-fucosidase is developmentally regulated.