The glycosylation enzyme α1,3 galactosyltransferase, which synthesizes the carbohydrate Galα1-3Galβ1-4GlcNAc-R, is active in non-primate mammals, prosimians and New World monkeys, but not in Old World monkeys, apes and humans. In this study, we have cloned and sequenced the enzyme expressed in a New World monkey, determined the exact size of the stem region and assessed the minimal size of catalytically active α1,3 galactosyltransferase (αl,3GT). Various primer sets were used in the polymerase chain reaction to generate cDNAs which coded for forms of α1,3GT with deletions at the N- or C-terminal domains. The cDNA was inserted into the expression vector pPROTA which contains the coding sequence for protein A, and subsequently transfected into COS cells. The soluble chimeric products (truncated enzyme and protein A) were harvested from the cell culture medium using IgG–Sepharose beads and assayed for enzymatic activity. As many as 67 amino acids could be truncated at the amino terminal region of the luminal portion of the enzyme without affecting its catalytic activity. Truncation of 68, 69 and 74 amino acids resulted in a 50, 75 and >95% loss in the in vitro catalytic actively, respectively. Introduction of a frameshift mutation which is characteristic of apes and human α1,3GT gene resulted in the complete loss of enzyme activity. Moreover, truncation of as few as three amino acids at the carboxyl end of α1,3GT resulted in complete loss of the catalytic activity. Replacement of the penultimate C-terminal Asn with Val also resulted in a major loss of enzyme activity. These findings indicate that the length of the stem region is 67 amino acids, the minimal size of an active enzyme is 285 amino acids, and that the amino acids at the carboxyl end of α1,3GT play an important role in maintaining the function of the enzyme. Similar characteristics were observed with murine αl,3GT. It is argued that a combination of the ape mutation and a regulatory mutation(s) which suppresses the transcription of this gene evolved to effectively prevent reversion of α1,3GT expression in apes and humans.