High concentrations of free C32 bis-homohopanoic acids (up to 433 μg/g dry wt) occur in microbial mats at methane seeps in anoxic Black Sea waters. These compounds show a strong preference for the ‘geological’ 17α(H),21β(H)- over the ‘biological’ 17β(H),21β(H)-configuration (αβ/ββ ratios up to 30.7) and indicate the potential formation of αβ-hopanoids in modern environments. Strong 13C-depletions (δ13C as low as −78.4‰ PDB) indicate an in situ generation of these hopanoids by biota involved in the anaerobic cycling of methane carbon. The inferred presence of hopanoids indigenous to a permanently anoxic marine environment is significant because these lipids are not known to occur in strictly anaerobic bacteria.