Values of Km were determined for three purified sialyltransferases and the corresponding recombinant enzymes. The enzymes were Gal beta 1-4GlcNAc alpha 2-6 sialyltransferase and Gal beta 1-3(4)GlcNAc alpha 2-3 sialyltransferase from rat liver; these enzymes are responsible for the attachment of sialic acid to N-linked oligosaccharide chains; and the Gal beta 1-3GalNAc alpha 2-3 sialyltransferase from porcine submaxillary gland that is responsible for the attachment of sialic acid to O-linked glycoproteins and glycolipids. A procedure for the large scale expression of active sialyltransferases from recombinant baculovirus-infected insect cells is described. For the liver enzymes values of Km were determined using rat and human asialo alpha 1 acid glycoprotein and N-acetyllactosamine as variable substrates; lacto-N-tetraose was also used with the Gal beta 1-3(4)GlcNAc alpha 2-3 sialyltransferases. Antifreeze glycoprotein was used as the macromolecular acceptor for the porcine enzyme. Values for Km were also determined using CMP-NeuAc as the variable substrate.