Membrane preparations from pig gastric mucosa were shown to transfer [14C]GlcNAc from UDP-[14C]GlcNAc to blood group A-negative porcine submaxillary mucin previously subjected to mild acid hydrolysis to remove terminal sialyl and fucosyl residues. O-Glycosyl oligosaccharides were removed from enzyme product by alkaline borohydride treatment and, after purification, were subjected to high resolution proton nuclear magnetic resonance spectroscopy and methylation analysis. Two trisaccharide products were detected: [14C]GlcNAc beta 1-3Gal beta 1-3GalNAcOH and Gal beta 1-3[( 14C]GlcNAc beta 1-6)GalNAcOH. We have previously reported the in vitro synthesis of the latter compound, a branched trisaccharide, by UDP-GlcNAc:Gal beta 1-3GalNAc-R (GlcNAc to GalNAc) beta 6-N-acetylglucosaminyltransferase from canine submaxillary glands. However, this is the first report of the in vitro synthesis of the linear trisaccharide GlcNAc beta 1-3Gal beta 1-3GalNAc. Pig gastric mucosal beta 3-N-acetylglucosaminyltransferase catalyzed the formation of this trisaccharide by incorporation of GlcNAc into the terminal Gal of Gal beta 1-3GalNAc-alpha-R when R was a polypeptide from either mucin or antifreeze glycoprotein, but not when R was o-nitrophenyl. We have previously reported the in vitro synthesis by pig gastric mucosa of the tetrasaccharide GlcNAc beta 1-3Gal beta 1-3(GlcNAc beta 1-6)GalNAc-alpha-R when R was o-nitrophenyl or benzyl. We show in this report that pig gastric mucosa can synthesize this tetrasaccharide in vitro when R is a polypeptide from either porcine submaxillary mucin or antifreeze glycoprotein. Pig gastric mucosa therefore contains a beta 6-N-acetylglucosaminyltransferase capable of converting Gal beta 1-3GalNAc-alpha-R to Gal beta 1-3(GlcNAc beta 1-6)GalNAc-alpha-R and one or more beta 3-N-acetylglucosaminyltransferases which can add GlcNAc in beta 1-3 linkage to a terminal Gal residue to form either GlcNAc beta 1-3Gal beta 1-3GalNAc-alpha-R or GlcNAc beta 1-3Gal beta 1-3(GlcNAc beta 1-6)GalNAc-alpha-R where R is the polypeptide backbone of either mucin or antifreeze glycoprotein.