Gammacerane is a triterpane compound first identified in the Green River shale. It is often found in sediments from hypersaline marine and nonmarine environments and has been suggested as an indicator of hypersalinity. However, recent studies show that gammacerane is derived from bacterivorous ciliates, which feed on green sulphur bacteria. This indicates that anaerobic ciliates living at or below the chemocline are important sources of gammacerane. The study shows that gammacerane is formed from tetrahymanol through sulphurisation and cleavage of C-S bonds, rather than through dehydration and hydrogenation as previously thought. This new pathway explains why gammacerane is often found in sediments deposited under hypersaline conditions but is not necessarily restricted to such deposits. It also explains why lacustrine deposits may contain abundant gammacerane, as most lakes in temperate climates are stratified during summer. The stable carbon isotopic compositions of gammacerane and lipids from primary producers and green sulphur bacteria in the Miocene Gessoso-solifera and Upper Jurassic Allgäu Formations indicate that gammacerane is derived from bacterivorous ciliates. This study concludes that gammacerane is an indicator of water column stratification, solving two current enigmas in gammacerane geochemistry. The study also shows that gammacerane is released from S-rich macromolecular aggregates by cleavage of C-S bonds during diagenesis and catagenesis. The results suggest that gammacerane is formed from tetrahymanol through sulphurisation and subsequent cleavage of C-S bonds, and that the precursor of gammacerane is likely a ketone, such as gammacer-3-one. The study also shows that the carbon isotopic composition of gammacerane is significantly enriched relative to lipids from primary producers, indicating that it is derived from ciliates that rely on green sulphur bacteria as a carbon source. This supports the idea that gammacerane is an indicator of water column stratification rather than hypersalinity. The study provides evidence for an alternative pathway for the formation of gammacerane from tetrahymanol, involving sulphurisation and cleavage of C-S bonds. This pathway is supported by the presence of S-bound gammacerane in kerogen and the release of gammacerane from S-rich macromolecular aggregates during artificial maturation. The study also shows that the carbon isotopic composition of gammacerane is consistent with its origin from ciliates that feed on green sulphur bacteria. This supports the conclusion that gammacerane is an indicator of water column stratification.Gammacerane is a triterpane compound first identified in the Green River shale. It is often found in sediments from hypersaline marine and nonmarine environments and has been suggested as an indicator of hypersalinity. However, recent studies show that gammacerane is derived from bacterivorous ciliates, which feed on green sulphur bacteria. This indicates that anaerobic ciliates living at or below the chemocline are important sources of gammacerane. The study shows that gammacerane is formed from tetrahymanol through sulphurisation and cleavage of C-S bonds, rather than through dehydration and hydrogenation as previously thought. This new pathway explains why gammacerane is often found in sediments deposited under hypersaline conditions but is not necessarily restricted to such deposits. It also explains why lacustrine deposits may contain abundant gammacerane, as most lakes in temperate climates are stratified during summer. The stable carbon isotopic compositions of gammacerane and lipids from primary producers and green sulphur bacteria in the Miocene Gessoso-solifera and Upper Jurassic Allgäu Formations indicate that gammacerane is derived from bacterivorous ciliates. This study concludes that gammacerane is an indicator of water column stratification, solving two current enigmas in gammacerane geochemistry. The study also shows that gammacerane is released from S-rich macromolecular aggregates by cleavage of C-S bonds during diagenesis and catagenesis. The results suggest that gammacerane is formed from tetrahymanol through sulphurisation and subsequent cleavage of C-S bonds, and that the precursor of gammacerane is likely a ketone, such as gammacer-3-one. The study also shows that the carbon isotopic composition of gammacerane is significantly enriched relative to lipids from primary producers, indicating that it is derived from ciliates that rely on green sulphur bacteria as a carbon source. This supports the idea that gammacerane is an indicator of water column stratification rather than hypersalinity. The study provides evidence for an alternative pathway for the formation of gammacerane from tetrahymanol, involving sulphurisation and cleavage of C-S bonds. This pathway is supported by the presence of S-bound gammacerane in kerogen and the release of gammacerane from S-rich macromolecular aggregates during artificial maturation. The study also shows that the carbon isotopic composition of gammacerane is consistent with its origin from ciliates that feed on green sulphur bacteria. This supports the conclusion that gammacerane is an indicator of water column stratification.