Heptosyltransferase II, encoded by the waaF gene of Escherichia coli, is a glycosyltransferase involved in the synthesis of the inner core region of lipopolysaccharide. The gene was subcloned from plasmid pWSB33 [Brabetz, W., Müller-Loennies, S., Holst, O. & Brade, H. (1997) Eur. J. Biochem. 247, 716-724] into a shuttle vector for the expression in the gram-positive host Corynebacterium glutamicum. The in vitro activity of the enzyme was investigated in comparison to that of heptosyltransferase I (WaaC) using as a source for the sugar nucleotide donor, ADP-LglyceroDmanno-heptose, a low molecular mass filtrate from a DeltawaaCF E. coli strain. Synthetic lipid A analogues varying in the acylation or phosphorylation pattern or both were tested as acceptors for the subsequent transfer of 3-deoxy-Dmanno-oct-2-ulosonic acid (Kdo) and heptose by successive action of Kdo transferase (WaaA), heptosyltransferase I (WaaC) and heptosyltransferase II (WaaF). The reaction products were characterized after separation by TLC and blotting with monoclonal antibodies specific for the acceptor, the intermediates and the final products.