The eDNA encoding the marmoset New World Monkey a l ,3 galactosyltransferase (al,3 GT) enzyme was cloned from a eDNA library of the B95.8 cell line and sequenced. To determine the minimal size necessary to retain catalytic activity a series of deletions were made in the coding region using specific sets of PCR primers. The truncated products were expressed as fusion proteins with staphylococcal protein A using the vector pPROTA. Up to 68 amino acids downstream of the transmembrane domain could be removed without decreasing the ability of the enzyme to transfer [3H] galactose to N-acetyllactosamine acceptor. The enzyme was remarkably sensitive, however, to truncations at the Cterminus, as removal of only two amino acids was sufficient to decrease activity of the enzyme by 95%. A comparison of the wild-type enzyme activity with that of a1,3 GT truncated by 64 amino acids downstream of the transmembrane domain revealed a similar rate of enzyme activity and a similar specificity for various carbohydrate acceptors, including a low but detectable utilization of galactose and N-acetylglucosamine. These results suggest an important role for amino acids at the C-terminus, either in catalysis or in maintenance of proper protein conformation, and define the length of the stem region of the enzyme, which extends the catalytic domains into the lumen of the Golgi apparatus, as 68 amino acids beyond the transmembrane domain.