In the genome sequence of Aspergillus niger CBS 513.88, three genes were identified with high similarity to fungal α-amylases. The protein sequences derived from these genes were different in two ways from all described fungal α-amylases: they were predicted to be glycosylphosphatidylinositol anchored, and some highly conserved amino acids of enzymes in the α-amylase family were absent. We expressed two of these enzymes in a suitable A. niger strain and characterized the purified proteins. Both enzymes showed transglycosylation activity on donor substrates with α-(1,4)- glycosidic bonds and at least five anhydroglucose units. The enzymes, designated AgtA and AgtB, produced new α-(1,4)-glycosidic bonds and therefore belong to the group of the 4-α-glucanotransferases (EC 220.127.116.11). Their reaction products reached a degree of polymerization of at least 30. Maltose and larger maltooligosaccharides were the most efficient acceptor substrates, although AgtA also used small nigerooligosaccharides containing α-(1,3)-glycosidic bonds as acceptor substrate. An agtA knockout of A. niger showed an increased susceptibility towards the cell wall-disrupting compound calcofluor white, indicating a cell wall integrity defect in this strain. Homologues of AgtA and AgtB are present in other fungal species with α-glucans in their cell walls, but not in yeast species lacking cell wall α-glucan. Possible roles for these enzymes in the synthesis and/or maintenance of the fungal cell wall are discussed. Copyright © 2007, American Society for Microbiology. All Rights Reserved.