Among mucus-secreting cells, the gastric gland mucous cells, Brunner’s glands, accessory glands of pancreaticobiliary tract, and pancreatic ducts exhibiting gastric metaplasia are unique in that they express class III mucin identified by paradoxical Con A staining composed of periodate oxidation, sodium borohydride reduction, Con A, and horseradish peroxidase reaction. Recently it was shown that these mucous cells secrete glycoproteins having GlcNAcα1→4Galβ→R at nonreducing terminals of the carbohydrate moieties. Herein we describe the expression cloning of a cDNA encoding a human α1,4-N-acetylglucosaminyltransferase (α4GnT), a key enzyme for the formation of GlcNAcα1→4Galβ1→R. COS-1 cells were thus cotransfected with a stomach cDNA library and a leukosialin cDNA. Transfected COS-1 cells were screened by using monoclonal antibodies specific for GlcNAcα1→4Galβ→R and enriched by fluorescence-activated cell sorting. Sibling selection of recovered plasmids resulted in a cDNA clone that directs the expression of GlcNAcα1→4Galβ→R. The deduced amino acid sequence predicts a type II membrane protein with 340 amino acids, showing no significant similarity with any other proteins. The α4GnT gene is located at chromosome 3p14.3, and its transcripts are expressed in the stomach and pancreas. An in vitro GlcNAc transferase assay by using a soluble α4GnT revealed that α1,4-linked GlcNAc residues are transferred most efficiently to core 2 branched O-glycans (Galβ1→4GlcNAcβ1→6(Galβ1→3)GalNAc), forming GlcNAcα1→4Galβ1→4GlcNAcβ1→6(GlcNAcα1→4Galβ1→3)GalNAc. Transfection of α4GnT cDNA into gastric adenocarcinoma AGS cells produced class III mucin, indicating that α4GnT is responsible for the formation of class III Con A reactivity. These results indicate that the α4GnT is a glycosyltransferase that forms α1,4-linked GlcNAc residues, preferentially in O-glycans.