Peptides containing a triprolyl sequence carboxyl to a threonine residue can be O-glycosylated by a crude Triton x-100 extract of porcine submaxillary glands (Young, J. D., Tsuchiya, D., Sandlin, D. E., and Holroyde, M. J. (1979) Biochemistry 18, 4444-4448). In the present paper, we have studied the characteristics of the O-glycosylating enzyme, UDP-N-acetylgalactosamine: polypeptide N-acetylgalactosamine transferase, from a membrane extract of bovine submaxillary glands using 11 synthetic peptide substrates in which the Thr-Pro-Pro-Pro was varied. The effect of these changes was measured by determining the apparent Km and Vmax values of the substrates. The studies thus far reveal: threonine cannot be glycosylated without a carboxyl triprolyl sequence; the alpha amino acid group of the threonine must be blocked; the nature of the group NH2-terminal to the threonine affects the kinetics of the reaction; and one residue can be between the threonyl and the triprolyl sequence. The triprolyl sequence in a protein may be an important signal for O-glycosylation.