Using a series of relevant substrates, connective tissue of the snail Lymnaea stagnalis was shown to contain beta 1-2 xylosyltransferase (beta 2Xyl-T), beta 1-2 N-acetylglucosaminyltransferase I (beta 2GlcNAc-T I), and beta 1-2 N-acetylglucosaminyltransferase II (beta 2GlcNAc-T II) activities. These enzymes are probably involved in the biosynthesis of the N-linked carbohydrate chains, like those present in hemocyanin. The products formed by incubation of GlcNAc beta 1-2Man alpha 1-6(GlcNAc beta 1-2Man alpha 1-3)Man beta 1-R [where R = -4GlcNAc beta 1-4GlcNAc or O-(CH2)7CH3] with UDP-Xyl and connective tissue microsomes have been purified and characterized by 1H-NMR spectroscopy in conjunction with methylation analysis to be GlcNAc beta 1-2Man alpha 1-6(GlcNAc beta 1-2Man alpha 1-3)(Xyl beta 1-2)Man beta 1-R. Substrate specificity studies focused on connective tissue beta 2Xyl-T show that the minimal structure requirements are fulfilled in GlcNAc beta 1-2Man alpha 1-3Man beta 1-O-(CH2)7CH3. The enzyme activity can therefore be characterized as UDP-Xyl:Glc-NAc beta 1-2Man alpha 1-3Man beta-R (Xyl to Man beta) beta 1-2 xylosyltransferase. In substrate-specificity studies directed to connective tissue beta 2GlcNAc-T I, it could be demonstrated that the enzyme is active towards acceptors having at the minimum a Man alpha 1-3Man beta-R sequence, and that introduction of a beta Xyl residue at C2 of beta Man totally abolishes the enzyme activity. Xylose-containing oligosaccharides are not acceptors for beta 2GlcNAc-T I. In combination with the substrate specificity of beta Xyl-T, this shows that in snail connective tissue beta 2GlcNAc-T I must act before beta 2Xyl-T. The connective tissue beta 2GlcNAc-T II activity follows the earlier established biosynthetic routes. Based on the substrate specificities of the various connective tissue glycosyltransferases known so far, and the structures isolated from L. stagnalis hemocyanin, a partial biosynthetic scheme for N-glycosylation in snail connective tissue is proposed.