Lipopolysaccharide of Haemophilus influenzae contains a single 3-deoxy-D-manno-octulosonic acid (Kdo) residue, linked to the 6' position of lipid A. In Escherichia coli and related organisms, a Kdo disaccharide is attached to lipid A. In previous studies, we cloned the gene (kdtA) encoding the E. coli Kdo transferase and demonstrated that homogeneous preparations of KdtA polypeptide catalyzed the attachment of both Kdo groups to the precursor, lipid IVA. E. coli KdtA produced only traces of mono-glycosylated product. We now show that a single Kdo is transferred to lipid IVA in extracts of H. influenzae. The mono-functional Kdo transferase of H. influenzae is membrane-bound, and the reaction is dependent upon a CMP-Kdo-generating system, as in E. coli. The specific activity of Kdo transfer to lipid IVA is 0.5-1 nmol/min/mg in H. influenzae membranes. Utilizing solubilized H. influenzae membranes, milligram quantities of Kdo-lipid IVA were prepared for analysis. Matrix-assisted laser desorption/ionization mass spectrometry revealed a parent ion (M - H)- at m/z 1626.0, consistent with the addition of a single Kdo moiety. Like lipid IVA, Kdo-lipid IVA was an excellent substrate for the bi-functional Kdo transferase of E. coli. In membranes of H. influenzae, but not E. coli, Kdo-lipid IVA was further phosphorylated in the presence of ATP, yielding a mono-phosphorylated Kdo-lipid IVA with a parent ion (M - H)- at m/z 1703.9. The identification of the mono-functional H. influenzae Kdo transferase, which is encoded by a KdtA homologue that displays 50% identity to its E. coli counterpart, should facilitate the mechanistic dissection of more complex multi-functional Kdo transferases, like those of E. coli and Chlamydia trachomatis.