Mucin type O-glycosylation is a widespread modification of eukaryotic proteins. The transfer of N-acetylgalactosamine to selected serine or threonine residues is catalyzed by a family of polypeptide N-acetylgalactosaminyltransferases localized in the Golgi apparatus. The most abundant elongation of O-glycans is the addition of a beta1-3 linked galactose by the core-1 beta1-3 galactosyltransferase (core-1 beta3GalT), thereby building the T-antigen or core-1 structure Gal(beta1-3)GalNAc(alpha1-O). We have isolated four Drosophila melanogaster cDNAs encoding proteins structurally similar to the human core-1 beta3GalT enzyme and expressed them as FLAG-tagged proteins in Sf9 insect cells. The identity of these D. melanogasterbeta3GalT enzymes with a core-1 beta3GalT activity was confirmed by utilization of MUC5AC mucin derived O-glycopeptide acceptors. In addition to the core-1 beta3GalT activity toward O-glycoprotein substrates, one member of this enzyme family showed a strong activity towards glycolipid acceptors, thereby building the core-1 terminated Nz6 glycosphingolipid. Transcripts of the embryonically expressed core-1 beta3GalTs were found in the maternally deposited mRNA, in salivary glands and in the amnioserosa. The presence of multiple core-1 beta3GalT genes in D. melanogaster suggests an increased complexity of core-1 O-glycan expression, which is possibly related to multiple developmental and physiological functions attributable to this class of glycans.