Some members of the glycoprotein hormone family [luteinizing hormone (LH), thyroid-stimulating hormone (TSH), and free alpha subunit] bear unique asparagine-linked oligosaccharides with the terminal sequence SO4-Gal-NAc beta 1,4GlcNAc beta 1,2Man alpha, whereas other members [human chorionic gonadotropin (hCG) and follicle-stimulating hormone (FSH)] bear predominantly oligosaccharides terminating in the sequence sialic acid alpha-Gal beta 1,4GlcNAc beta 1,2Man alpha. We previously identified an N-acetylgalactosaminetransferase (GalNAc-transferase) in bovine pituitary membranes that specifically recognizes the alpha subunit peptide and adds GalNAc to the synthetic intermediate GlcNAc2Man3GlcNAc2. In the current study we demonstrate that bLH, hCG, hCG beta, hCG alpha, and FSH alpha are recognized by the pituitary GalNAc-transferase in vitro, whereas oFSH, hFSH, and hFSH beta are not (b-, h-, and o-indicate bovine, human, and ovine). The apparent Km values for addition of GalNAc to oligosaccharides on hCG alpha and hCG beta, 13.0 and 6.2 microM, respectively, are not altered by reduction and alkylation. Thus, recognition of the peptide determinant does not require maintenance of native tertiary structural features. In the presence of the recognition determinant the Km for addition of GalNAc to the intermediate GlcNAc2Man3GlcNAc2 is reduced from 1.2-2.6 mM to less than 13 microM. hFSH is not efficiently recognized by the GalNAc-transferase due to the absence of the recognition marker on hFSH beta and some degree of masking of the recognition marker on the alpha subunit when combined with FSH beta. Since recognition is directed at primary and possibly secondary structural features, it should be possible to determine which regions of the alpha and beta subunits are responsible for the specificity of the GalNAc-transferase.