The specificity of fetal calf liver and ovine and porcine submaxillary gland N-acetylgalactosaminide alpha 2 leads to 6 sialyltransferase was investigated with acceptors of low and high molecular weight containing O-glycosidically linked carbohydrate chains. It appeared that fetal calf liver microsomes were able to transfer sialic acid to C-6 of GalNAc in NeuAc(alpha 2 leads to 3)Gal(beta 1 leads to 3)GalNAc-R, in which the aglycon could be protein as well as p-nitrophenol (Nph). The substrates Gal(beta 1 leads to 3)GalNAc-R and GalNAc-R were inactive as acceptor with this enzyme source. Ovine and porcine submaxillary gland microsomes were both active with GalNAc-protein, Gal(beta 1 leads to 3)GalNAc-protein, NeuAc[alpha 2 leads to 3)Gal(beta 1 leads to 3)GalNAc-protein and NeuAc(alpha 2 leads to 3)Gal(beta 1 leads to 3)GalNAc alpha-Nph, but not with GalNAc alpha-Nph and Gal(beta 1 leads to 3)GalNAc alpha-Nph. The N-acetylgalactosaminide alpha 2 leads to 6 sialyltransferase which had been purified to homogeneity from porcine submaxillary gland [Sadler, J. E., Rearick, J. I. and Hill, R. L. (1979) J. Biol. Chem. 254, 5934-5941], was able to sialylate all three protein acceptors, but was virtually inactive with each of the three p-nitrophenyl glycosides. Our studies indicate that two N-acetylgalactosaminide alpha 2 leads to 6 sialtransferases exist acting on O-glycosidically linked carbohydrate chains. The first enzyme, present in fetal calf liver, has a narrow specificity with regard to the oligosaccharide structure, but shows no specificity for the aglycon. Based on its specificity this enzyme can be designated as an [alpha-N-acetylneuraminosyl 2 leads to 3-beta-galactosyl 1 leads to 3]-alpha-N-acetylgalactosaminide alpha 2 leads to 6 sialyltransferase. The second enzyme, present in porcine submaxillary gland, has an absolute requirement for protein as the aglycon. Once this condition is fulfilled, the enzyme is able to transfer sialic acid to each of the three oligosaccharide chains and thus the enzyme is an alpha-N-acetylgalactosaminylprotein alpha 2 leads to 6 sialyltransferase. The data also seem to suggest that ovine and porcine submaxillary gland microsomes contain, in addition to the latter enzyme activity, the alpha 2 leads to 6 sialyltransferase with the strict oligosaccharide specificity.