Stable carbon isotopes can provide insight into carbon cycling pathways in natural environments. We examined carbon isotope fractionations associated with a hyperthermophilic fermentative bacterium, Thermotoga maritima, and a thermophilic chemolithoautotrophic bacterium Persephonella marina. In T. maritima, phospholipid fatty acids (PLFA) are slightly enriched in ¹³C relative to biomass (ε = 0.1–0.8‰). However, PLFA and biomass are depleted in ¹³C relative to the substrate glucose by ~8‰. In P. marina, PLFA are 1.8–14.5‰ enriched in ¹³C relative to biomass, which suggests that the reversed tricarboxylic acid (TCA) cycle or the 3-hydroxypropionate pathway may be used for CO₂ fixation. This is supported by small fractionation between biomass and CO₂ (ε = -3.8‰ to -5.0‰), which is similar to fractionations reported for other organisms using similar CO₂ fixation pathways. Identification of the exact pathway will require biochemical assay for specific enzymes associated with the reversed TCA cycle or the 3-hydroxypropionate pathway.