The acceptor substrate specificity of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (GalNAc-transferase) was inferred from the amino acid sequences surrounding 196 O-glycosylation sites extracted from the National Biomedical Research Foundation Protein Database. When analyzed according to the cumulative enzyme specificity model (Poorman, R.A., Tomasselli, A.G., Heinrikson, R.L., and Kézdy, F.J. (1991) J. Biol. Chem. 266, 14554-14561) these data were found to be consistent with an enzymatic active site which interacts with an 8-amino-acid long segment of the substrate, spanning 3 amino acid residues preceding and 4 amino acid residues following the reactive serine or threonine. The model postulates independent interactions of the 8 amino acid moieties with their respective binding sites, designated as subsites P3 through P0 and P1' to P4'. High selectivity is expressed at all subsites toward serine, threonine, and proline. The inferred specificity was confirmed by in vitro bovine colostrum GalNAc-transferase-catalyzed glycosylation of unglycosylated proteins containing predicted sites for O-glycosylation and synthetic peptides designed to be GalNAc acceptors. In synthetic peptides the bovine colostrum GalNAc-transferase glycosylates threonine about 35 times faster than serine. Our results suggest that the specificity of the enzyme is not dependent on any particular secondary structure of the substrate but, rather, it is determined by the amino acids in the acceptor peptide segment as well as by the accessibility of this segment. It also appears likely that bovine colostrum GalNAc-transferase is able to catalyze in vivo the glycosylation of both threonine and serine residues.