The biosynthesis of HNK-1 carbohydrate is mainly regulated by two glucuronyltransferases (GlcAT-P and GlcAT-S) and a sulfotransferase (HNK-1 ST). To determine how the two glucuronyltransferases are involved in the biosynthesis of the HNK-1 carbohydrate, we prepared soluble forms of GlcAT-P and GlcAT-S fused with the IgG-binding domain of protein A and then compared the enzymatic properties of the two enzymes. Both GlcAT-P and GlcAT-S transferred glucuronic acid (GlcA) not only to a glycoprotein acceptor, asialoorosomucoid (ASOR), but also to a glycolipid acceptor, paragloboside. The activity of GlcAT-P toward ASOR was enhanced fivefold in the presence of sphingomyelin, but there were no effects on that of GlcAT-S. The activities of the two enzymes toward paragloboside were only detected in the presence of phospholipids such as phosphatidylinositol. Kinetic analysis revealed that the K(m) value of GlcAT-P for ASOR was 10 times lower than that for paragloboside. Furthermore, acceptor specificity analysis involving various oligosaccarides revealed that GlcAT-P specifically recognized N-acetyllactosamine (Galbeta1-4GlcNAc) at the nonreducing terminals of acceptor substrates. In contrast, GlcAT-S recognized not only the terminal Galbeta1-4GlcNAc structure but also the Galbeta1-3GlcNAc structure and showed the highest activity toward triantennary N-linked oligosaccharides. GlcAT-P transferred GlcA to NCAM about twice as much as to ASOR, whereas GlcAT-S did not show any activity toward NCAM. These lines of evidence indicate that these two enzymes have significantly different acceptor specificities, suggesting that they may synthesize functionally and structurally different HNK-1 carbohydrates in the nervous system.