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Lake sediments are important archives of continental climate history, and their lipid biomarker content can be exploited to reconstruct paleoenvironmental conditions. Branched glycerol dialkyl glycerol tetraethers (brGDGTs) are bacterial membrane lipids widely used in paleoclimate studies to reconstruct past temperature. However, major gaps still exist in our understanding of the environmental controls on in situ (i.e. aquatic) production in lake systems. In Lake Chala, a permanently stratified tropical crater lake in East Africa, we determined the concentrations and fractional abundances of individual brGDGTs along depth profiles of suspended particulate matter collected monthly from September 2013 to January 2015 and in settling particles collected monthly at 35 m water depth from August 2010 to January 2015 and compared these brGDGT distributions with those in surficial lake bottom sediments and catchment soils. We find that brGDGTs are primarily produced within the water column and that their concentrations and distributions vary greatly with depth and over time. Comparison with concentration–depth profiles of the monthly distribution and abundance of bacterial taxa, based on 16S rRNA gene amplicon sequencing and quantification, indicates that Acidobacteria are likely not the main producers of brGDGTs in Lake Chala. Shallowing of the oxic–anoxic boundary during seasonal episodes of strong water column stratification promoted production of specific brGDGTs in the anoxic zone. BrGDGT distributions in the water column do not consistently relate with temperature, pH, or dissolved-oxygen concentration but do respond to transitions between episodes of strong stratification and deep (but partial) lake mixing, as does the aquatic bacterial community. Hence, the general link between brGDGT distributions and temperature in brGDGT-based paleothermometry is more likely driven by a change in bacterial community composition than by membrane adaptation of specific members of the bacterial community to changing environmental conditions. Although temperature is not the principal driver of distributional changes in aquatic brGDGTs in this system, at least not during the 17-month study period, abundance-weighted and time-integrated averages of brGDGT fractional abundance in the 53-month time series of settling particles reveal systematic variability over longer timescales that indirectly relates to temperature. Thus, although we do not as yet fully understand the drivers of modern-day brGDGT fluxes and distributions in Lake Chala, our data do support the application of brGDGT paleothermometry to time-integrated archives such as sediments. Highlights. BrGDGTs in the tropical African lake Chala are produced in situ. Acidobacteria are not the dominant source of aquatic brGDGTs. Stratification and mixing drive aquatic brGDGT production and their signature.