A number of N- and C-terminal deletion and point mutants of bovine beta-1,4 galactosyltransferase (beta-1,4GT) were expressed in E. coli to determine the binding regions of the enzyme that interact with N-acetylglucosamine (NAG) and UDP-galactose. The N-terminal truncated forms of the enzyme between residues 1-129, do not show any significant difference in the apparent Kms towards NAG or linear oligosaccharide acceptors e.g. for chitobiose and chitotriose, or for the nucleotide donor UDP-galactose. Deletion or mutation of Cys 134 results in the loss of enzymatic activity, but does not affect the binding properties of the protein either to NAG- or UDP-agarose. From these columns the protein can be eluted with 15 mM NAG and 50 mM EDTA, like the enzymatically active protein, TL-GT129, that contains residues 130-402 of bovine beta-1,4GT. Also the N-terminus fragment, TL-GT129NAG, that contains residues 130-257 of the beta-1,4GT, binds to, and elutes with 15 mM NAG and 50 mM EDTA from the NAG-agarose column as efficiently as the enzymatically active TL-GT129. Unlike TL-GT129, the TL-GT129NAG binds to UDP-columns less efficiently and can be eluted from the column with only 15 mM NAG. The C-terminus fragment GT-257UDP, containing residues 258-402 of beta-1,4GT, binds tightly to both NAG- and UDP-agarose columns. A small fraction, 5-10% of the bound protein, can be eluted from the UDP-agarose column with 50 mM EDTA alone. The results show that the binding behaviour of N- and C-terminal fragments of beta-1,4GT towards the NAG- and UDP-agarose columns differ, the former binds preferentially to NAG-columns, while the latter binds to UDP-agarose columns via Mn2+.