A specific and fast method for the determination of N-acetylglucosaminyltransferase III, IV and V activity in one assay is described. The method is based on the separation by HPLC of the three tranferase products formed from the common acceptor oligosaccharide substrate GlcNAcβ1[RIGHTWARDS ARROW]2Manα1[RIGHTWARDS ARROW]3(GlcNAcβ1[RIGHTWARDS ARROW]2Manαl[RIGHTWARDS ARROW]6)Manβ1[RIGHTWARDS ARROW]4GlcNAc. Assays are not interfered with by substances that result from enzymatic or chemical breadkdown of the donor substrate UDP-[14C]GlcNAc. Using this assay system N-acetylglucosaminyltransferase III, IV and V activities were estimated in Novikoff ascites tumour cells, mouse lymphoma BW 5147 cells and hen oviduct.