The production of bacteriohopanepolyols (BHPs) is widespread in many different groups of prokaryotes; however, unsaturated components are less common except amongst the acetic acid bacteria. Here we describe the characterisation of mono- (Delta(6) or Delta(11)) and diunsaturated (Delta(6,11)) bacteriohopanetetrols isolated from the acetic acid bacterium Gluconacetobacter xylinus (formerly Acetobacter aceti ssp. xylinum) by atmospheric pressure chemical ionisation ion trap mass spectrometry (APCI-MS(n)). APCI-MS(2) spectra are compared with equivalent electron ionisation (EI) spectra and differences in fragmentation pathways are discussed. Having established characteristic spectral features for a range of unsaturated BHPs we now have the ability to rapidly detect the presence of unsaturated BHPs in both natural environmental samples (soils, sediments, water columns) as well as in microbial cultures.