RATIONALE Over the last decade, the high lateral resolution and imaging capabilities of time-of-flight secondary ion mass spectrometry (ToF-SIMS) have increasingly stimulated interest in studying organic molecules in complex environmental materials. However, unlike with the established mass spectrometric techniques, the use of ToF-SIMS in the biogeosciences is still hampered by a lack of reference spectra of the relevant biomarker compounds. Here we present and interpret ToF-SIMS reference spectra of ten different cyclic lipids that are frequently used as biological tracers in ecological, organic geochemical and geobiological studies. METHODS Standard compounds of α,β,β-(20R, 24S)-24-methylcholestane, (22E)-ergosta-5,7,22-trien-3β-ol, 17α(H),21β-(H)-30-norhopane, hope-17(21)-ene, hop-22(29)-ene, 17β(H),21β(H)-bacteriohopane-32,33,34,35-tetrol, 17β(H), 21β(H)-35-aminobacteriohopane-32,33,34-triol, α-tocopherol, β,β-carotene, chlorophyll a, and cryosections of microbial mats and a fungus were analyzed using a ToF-SIMS instrument equipped with a Bi 3+ cluster ion source. RESULTS The spectra obtained from the standard compounds showed peaks in the molecular weight range (molecular ions, protonated and deprotonated molecules, adduct ions) and diagnostic fragment ion peaks in both, positive and negative ion modes. For the cyclic hydrocarbons, however, the positive ion mode spectra typically showed more and stronger characteristic peaks than the negative ion mode spectra. Using real world samples the capability of ToF-SIMS to detect and image selected compounds in complex organic matrices was tested. 17β(H),21β(H)-35- Aminobacteriohopane-32,33,34-triol, carotene and chlorophyll a were successfully identified in cryosections of microbial mats, and the distribution of ergosterol was mapped at μm resolution in a cryosection of a fungus (Tuber uncinatum). CONCLUSIONS This study further highlights the utility of ToF-SIMS for the identification and localization of lipids within environmental samples and as a technique for biomarker-related research in organic geochemistry and geobiology.