Unlike classical β-1,4-galactosyltransferase (β-1,4-GalT I), β-1,4-GalT V (formerly IV**) has little activity towards 1 mM N-acetylglucosamine. The human β-1,4-GalTs I and V were expressed individually in Sf-9 cells by transfection of the full coding sequences, and their N-acetyllactosamine synthetase activities were determined towards different N-acetylglucosamine concentrations. Kinetic studies using the cell homogenates as an enzyme source revealed that β-1,4-GalTs I and V possess Km values of 0.6 mM and 33 mM towards N-acetylglucosamine, and of 48 μM and 41 μM towards UDP-Gal, respectively. No significant inhibition of N-acetyllactosamine synthesis with α-lactalbumin was observed for β-1,4-GalT V but the significant inhibition with α-lactalbumin was observed for β-1,4-GalT I.