Endo-alpha-N-acetyl-D-galactosaminidase from Diplococcus pneumoniae was shown to have transglycosylation and transfer reaction (reversed hydrolysis) activities. Treatment of asialoglycoproteins having Gal beta 1----3GalNAc alpha 1----Ser/Thr linkages with enzyme preparations containing glycerol resulted in formation of nonreducing trisaccharides. The structure of the main trisaccharide (approximately 80%) was deduced to be Gal beta 1----3GalNAc alpha 1----1(3)-glycerol by analysis of sugar composition and the results of exoglycosidase treatment and periodate oxidation. The ability of the endoglycosidase to catalyze transfer of Gal beta 1----3GalNAc to various acceptors was also demonstrated by incubation of the enzyme with the disaccharide and the test compound. The following were found to show acceptor activity: glycerol, Tris, p-nitrophenol, threonine, serine, D-glucose, D-galactose, D-fucose, and 6-O-methylgalactose. Transfer to the primary hydroxyl groups of glycerol and hexoses appears to be favored since the major glycerol product was 1(3)-substituted and transfer to D-fucose and 6-O-methyl-D-galactose was less than that to D-galactose. In order to avoid spurious results, it is necessary to carry out incubations with this enzyme in the absence of glycerol and other hydroxy compounds. The potential use of this endoglycosidase in the synthesis of glycosides is indicated by our studies.