The use of electrospray Ionization (ESI) tandem mass spectrometry (MS/MS) for the structural determination of the lipid A components of the psychrophilic bacterium Pseudoalteromonas haloplanktis TAC 125 is reported. The lipid A contains the classical bisphosphorylated beta-(1' --> 6)-linked D-glucosamine disaccharide with 3-hydroxydodecanoyl residues (12 : 0 (3-OH)) linked both as esters and amides to 2', 3' (distal glucosamine) and 2, 3 positions (proximal glucosamine) of the sugar backbone. The hydroxyl of 12 : 0 (3-OH) fatty acid linked at the 3' position is esterified by a dodecanoyl residue (12 : 0). In addition to the pentaacyl component, a minor tetraacyl lipid A, lacking the acyl residue at position 3, was also found in the lipid A fraction. The advantage of this MS technique for the investigation of the intra-ring fragmentation, which is useful for the determination of fatty acyl residue distribution on each glucosamine unit, is emphasized. Copyright 2002 John Wiley & Sons, Ltd.