Lipid A isolated from the Rickettsia typhi lipopolysaccharide (LPS) was investigated for its composition and structure using chemical analyses, gas chromatography-mass spectrometry (GC-MS), and electrospray ionization (ESI) combined with the tandem mass spectrometry (MS/MS). Our studies revealed a noticeable compositional and structural heterogeneity of lipid A with respect to the content of phosphate groups and the degree of acylation. It appeared that at least two molecular species were present in lipid A. The major species represented the hexaacyl lipid A consisting of the β-(1--> 6)-linked D-glucosamine (GlcN) disaccharide backbone carrying two phosphate groups. One of them was linked to the glycosidic hydroxyl group of the reducing GlcN I and the other was ester linked to the O-4´ position of the non-reducing GlcN II. The primary fatty acids consisted of two 3-hydroxytetradecanoic [C14:0(3-OH)] and two 3-hydroxyhexadecanoic [C16:0(3-OH)] acids. The former were ester- and the latter amide-linked to both GlcN. Two secondary fatty acids were represented by the octadecanoic (C18:0) and hexadecanoic (C16:0) acids that were ester-linked at the N-2´ and O-3´ positions, respectively. In the minor lipid A species, ester-linked C18:0 was substituted by C16:0 at the C16:0(3-OH) of GlcN II. The R. typhi lipid A resembles structurally the classical forms of enterobacterial lipids A with high endotoxicity.