A β1,6N-acetylglucosaminyltransferase (β1-6GnT) responsible for the formation of the β1,6-branched poly-N-acetyllactosamine structure has been purified 210,000-fold in 2.4% yield from a homogenate of hog small intestine by successive column chromatographies involving CM-Sepharose FF, Ni2+- chelating Sepharose FF, and UDP-hexanolamine-agarose, using an assay wherein pyridylaminated lacto-N-neotetraose (Galβ1-4GlcNAcβ1-3Galβ1-4Glc-PA) was used as an acceptor substrate, and the reaction product was Galβ1-4Glc- NAcβ1-3(GlcNAcβ1-6)Galβ1-4Glc-PA. The apparent molecular weight of the purified enzyme was 76,000 under nonreducing conditions. The enzyme has a pH optimum at 7.0 and has no requirement for any divalent metal ions. The K(m) values for pyridylaminated lacto-N-neotetraose and UDP-GlcNAc were 0.96 and 2.59 mM, respectively. For its activity, this enzyme was shown to have an absolute requirement of at least a complete LacNAc (LacNAc = Galβ1-4GlcNAc) residue bound to position 3 of the acceptor Gal residues, i.e. it is capable of acting only on the Gal residues of internal LacNAc units. The data strongly suggest that this enzyme could be involved in generating branches to central positions of preformed as well as growing polylactosamine chains, but not in synthesizing the distal branches to growing polylactosamine chains.