Ladderane lipids, containing three or five linearly concatenated cyclobutane moieties, are considered to be unique biomarkers for the process of anaerobic ammonium oxidation, an important link in the oceanic nitrogen cycle. Due to the thermal lability of the strained cyclobutane moieties, the ladderane lipids are difficult to analyze by gas chromatography. A method combining high-performance liquid chromatography coupled to positive ion atmospheric pressure chemical ionization tandem mass spectrometry (HPLC/APCI-MS/MS) was developed for the analysis of the most abundant ladderane lipids, occurring as fatty acids and ether-bound to glycerol. Detection was achieved by selective reaction monitoring of four specific fragmentations per ladderane lipid. Detection limits of 30–35 pg injected on-column and a linear response (r2 > 0.99) over nearly 3 orders of magnitude were achieved for all compounds. Using this method, these unique ladderane lipids were for the first time identified in a surface sediment from the Gullmarsfjorden, in concentrations ranging from 1.1–5.5 ng/g for the ladderane fatty acids and of 0.7 ng/g for the monoether. It is foreseen that this method will allow the investigation of the occurrence of anaerobic ammonium oxidation in natural settings in much greater detail than before. Copyright © 2006 John Wiley & Sons, Ltd.