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Different pathways for autotrophic CO 2 fixation can be recognized by the presence of genes for their specific key enzymes. On this basis, (meta)genomic, (meta)transcriptomic, or (meta)proteomic analysis enables the identification of the role of an organism or a distinct pathway in primary production. However, the recently discovered variant of the reductive tricarboxylic acid (rTCA) cycle, the reverse oxidative tricarboxylic acid (roTCA) cycle, lacks unique enzymes, a feature that makes it cryptic for bioinformatics analysis. This pathway is a reversal of the widespread tricarboxylic acid (TCA) cycle. The functioning of the roTCA cycle requires unusually high activity of citrate synthase, the enzyme responsible for citrate cleavage, as well as elevated CO 2 partial pressures. Here, we present a detailed description of the protocol we used for the identification of the roTCA cycle in members of Desulfurellaceae . First, we describe the anaerobic cultivation of Desulfurellaceae at different CO 2 concentrations with a method that can be adapted to the cultivation of other anaerobes. Then, we explain how to measure activities of enzymes responsible for citrate cleavage, malate dehydrogenase reaction, and the crucial carboxylation step of the cycle catalyzed by pyruvate synthase in cell extracts. In conclusion, we describe stable isotope experiments that allow tracking of the roTCA cycle in vivo , through the position-specific incorporation of carbon-13 into amino acids. The label is provided to the organism as 13 CO 2 or [1- 13 C]glutamate. The same key methodology can be used for the reliable evaluation of the functioning of the roTCA cycle in any organism under study. This pathway is likely to participate, completely unseen, in the metabolism of various microorganisms. Graphic abstract., Competing Interests: Competing interestsThe authors have no conflicts of interest or competing interests., (Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC.)