Previous work has shown that odd-numbered oligosaccharides containing nonreducing terminal, non-sulfated N-acetylgalactosamine (GalNAc) or 6-sulfated GalNAc are excellent acceptors for enzymic addition of glucuronic acid (GlcA). However, the presence of a 4-sulfated GalNAc group blocks further addition. We have now used even-numbered oligosaccharides (a mixture of 4-sulfated, 6-sulfated, and non-sulfated) as acceptors of [3H]GalNAc to investigate the effect of sulfate residues on the GalNAc in the penultimate position. 3H-Labeled oligosaccharides were partially degraded with chondroitin AC lyase. The labeled trisaccharides, consisting of the added [3H]GalNAc and the nonreducing terminal disaccharides of the oligosaccharide acceptors, were then characterized. Both non-sulfated and mono-sulfated 3H-trisaccharides were observed. However, the 3H-trisaccharides were shown by chromatography with prepared standards to be non-sulfated or to be sulfated only at the 6-position. Thus the oligosaccharides containing a 4-sulfated, penultimate GalNAc at the nonreducing end did not serve as acceptors for [3H]GalNAc. Microsomes from mastocytoma cells, which make only chondroitin 4-sulfate, exhibited the same substrate specificity for exogenous oligosaccharide acceptors as did microsomes from chick cartilage which makes chondroitin 6-sulfate.