Using a series of relevant substrates, connective tissue of the snail Lymnaea stagnalis was shown to contain β1‐2 xylosyltransferase (β2Xyl‐T), β1‐2 N‐acetylglucosaminyltransferase I (β2GlcNAc‐T I), and β1‐2 N‐acetylglucosaminyltransferase II (β2GlcNAc‐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 GlcNAcβ1‐2Manα1‐6(GlcNAcβ1‐2Manα1‐3)Manβ1‐R [where R = ‐4GlcNAcβ1‐4GlcNAc 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 GlcNAcβ1‐2Manα1‐6(GlcNAcβ1‐2Manα1‐3)(Xylβ1‐2)Manβ1‐R. Substrate specificity studies focused on connective tissue β2Xyl‐T show that the minimal structure requirements are fulfilled in GlcNAcβ1‐2Manα1‐3Manβ1‐O‐(CH2)7CH3. The enzyme activity can therefore be characterized as UDP‐Xyl: GlcNAcβ1‐2Manα1‐3Manβ‐R (Xyl to Manβ) β1‐2 xylosyltransferase. In substrate‐specificity studies directed to connective tissue β2GlcNAc‐T I, it could be demonstrated that the enzyme is active towards acceptors having at the minimum a Manα1‐3Manβ‐R sequence, and that introduction of a βXyl residue at C2 of βMan totally abolishes the enzyme activity. Xylose‐containing oligosaccharides are not acceptors for β2GlcNAc‐T I. In combination with the substrate specificity of β2Xyl‐T, this shows that in snail connective tissue β2GlcNAc‐T I must act before β2Xyl‐T. The connective tissue β2GlcNAc‐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.