Mitogenic and polyclonal B cell activation (PBA) activities of 16 synthetic compounds related to the nonreducing D-glucosamine (GlcN-II) subunit of lipid A were investigated. Among compounds possessing the GlcN backbone, a 4-O-phosphorylated GlcN derivative carrying N-linked 3-tetradecanoyloxytetradecanoyl [C14-O-(C14)] and 3-O-linked tetradecanoyl (C14) groups, GLA-27, expressed the highest degree of both activities. Omission of the 3-O-linked C14 group from GLA-27 and transfer of the C14 group to the C-6 position induced critical changes in expression of activities. Both 4-O-phosphorylated compounds carrying an N-linked C14 or 3-hydroxytetradecanoyl (C14OH) group instead of the C14-O-(C14) group in GLA-27 showed no detectable activity. Substituting a 3-O-linked C14 group in GLA-27 for the C14-O-(C14) group also markedly decreased mitogenic and PBA activities. Change of phosphorylation site from the C-4 to the C-6 position and bisphosphorylation at the C-4 and C-6 positions induced somewhat weak depression. Much weaker activities were observed in a compound carrying N-linked 3-dodecanoyloxydodecanoyl [C12-O-(C12)] and 3-O-dodecanoyl (C12) as fatty acid substituents. No detectable activity was seen in a compound carrying N-linked 3-hexadecanoyloxyhexadecanoyl [C16-O-(C16)] and 3-O-hexadecanoyl (C16), indicating that the most suitable carbon chain length for expressing the activities is C14. Regarding structural change of the GlcN backbone, a 1-deoxy derivative of GLA-27 exhibited stronger activity than did GLA-27 itself. Mitogenic and PBA activity of GLA-27 were stronger than those of lipid X, which corresponds to the reducing D-GlcN (GlcN-I) subunit of Escherichia coli lipid A and is a 1-O-phosphorylated GlcN derivative carrying N- and 3-O-linked C14OH groups. These results indicate that N-linked acyloxyacyl and 3-O-linked acyl groups and phosphorylation are critical for expressing both mitogenic and PBA activities.