The original B transferase construct possessed A and expressed B antigens. In the in vitro enzymatic assays, weak activity of A transferase was also detected as previously reported (Greenwell et al., 1986). When it was replaced with smaller G, the construct expressed both A and B antigens. When it was replaced with slightly larger S or C, the constructs still exhibited strong B antigen expression. When it was replaced with larger N or T, the constructs exhibited decreased B antigen expression. When the N and D constructs were compared with the glutamine (Q) and glutamic acid (E) constructs, the expression of B antigen was stronger with the N and D constructs than the Q and E constructs. Similarly, when the N construct was compared with the D construct or when the Q construct was compared with the E construct, the expression of the B antigen was stronger with the N construct than with the D construct and also stronger with the Q construct than with the E construct. Taken together, it was concluded that the size and charge of the amino acid residue at codon 268 was crucial in determining the activity and specificity of the glycosyltransferases. Because the results differed between the constructs with the A transferase backbone and those with the B transferase backbone, it was also shown that not only codon 268, but also amino acid substitutions at other 3 locations, are important, with codon 266 most likely being of primary importance.