Amino acid sequence alignment of human alpha1, 3/4-fucosyltransferases (FucTs) demonstrates that three highly conserved Lys residues are present in the catalytic domain of FucTs III, IV, V, and VI. Two of these sites are conserved in FucT VII, with the third located within the alpha1,3-FucT motif as a conservative change to Arg at position 223. Site-directed mutagenesis experiments were conducted to change Lys255 of FucT V (equivalent to Arg223 of FucT VII) to either Arg255 or Ala255. Enzyme assays demonstrate that the FucT V K255R mutant has a 34-fold lower specific activity than native FucT V and that the K255A mutant is inactive. Site-directed mutagenesis of FucT VII was also conducted to change Arg223 to Lys223 for analysis of the effect on enzyme kinetic parameters. No differences in acceptor specificities or Km values for either substrate were observed between native FucT VII and the R223K mutant; however, the purified R223K mutant enzyme had a 2-fold increased specific activity compared with purified native FucT VII. No change in GDP-fucose-protectable pyridoxal-P/NaBH4 inactivation was observed for native or mutant FucT V or VII, further supporting the absence of involvement of this residue in sugar nucleotide binding. The results indicate that a basic residue in this position is required for enzyme activity, with a Lys residue providing higher intrinsic activity. The lack of influence of this site on substrate binding parameters and its location within the alpha1,3-FucT motif suggest that at least some of the residues within this motif are involved in catalysis rather than substrate binding.