Histo-blood group A transferase produces A antigens and transfers GalNAc to the acceptor substrate, H structures of glycolipids and glycoproteins. B transferase transfers galactose in place of GalNAc to the same acceptor substrate to synthesize B antigens. We have previously identified four amino acid substitutions between human A and B transferases. Out of these four, substitutions at the last two positions (codons 266 and 268) were found to be crucial for the different donor nucleotide-sugar specificities between A and B transferases as analyzed by gene transfer of chimeric A-B transferase genes. In the present study, we have in vitro mutagenized codon 268 of these two transferase cDNA expression constructs (glycine and alanine in A and B transferases, respectively) and produced substitution constructs with every possible amino acid residue at this position. We examined the activity and specificity of each construct by gene transfer followed by immunodetection of A and B antigens and in vitro enzymatic assay. Amino acid substitution constructs on the A transferase backbone with alanine, serine, and cysteine expressed enzymes with A and B transferase activities. Weak A activity was detected with histidine and phenylalanine constructs while weak B activity was detected with asparagine and threonine constructs. All the other amino acid substitutions at codon 268 on the A transferase backbone showed neither A nor B activity. The glycine construct on the B transferase backbone expressed both A and B transferase activities. Some substitution constructs on the B transferase backbone maintained B activity while some other substitutions abolished the activity. These results show that the side chain of the amino acid residue at 268 of the human A and B transferases is responsible for determining both activity and nucleotide-sugar donor substrate specificity and strongly suggest its direct involvement in the recognition of and binding to the sugar moiety of the nucleotide-sugars.