Motivated by the finding that Pseudomonas knackmussii B13 but not Rhodococcus opacus 1CP grows in the absence of externally provided CO2, we investigated the assimilation of 13CO2 into active cells cultivated with non-labelled glucose as sole energy substrate. 13C found in the bulk biomass indicated a substantial but different CO2 assimilation by Pseudomonas and Rhodococcus, respectively (3500‰ and 2600‰). Cellular fatty acids were labelled from −15‰ to 470‰ and amino acids from 500‰ to 24 000‰ demonstrating clear differences between various compound classes. ‘You are what you eat plus 1‰’ is therefore only valid for the average bulk C without specific isotope signature deviation of the external CO2 or carbonates. Odd-numbered and 10-methyl fatty acids, which are much more abundant in Rhodococcus or other Gram-positive bacteria, were up to fivefold higher enriched in 13C relative to the Pseudomonas fatty acids. A high-level growth-phase-independent, labelling of the oxaloacetate-derived amino acids indicated heterotrophic CO2 fixation by anaplerotic reactions known to replenish the tricarboxylic acid cycle. Although both strains assimilate CO2 via similar general pathways, Rhodococcus depends to a much higher extent on carboxylations reactions with external CO2 owing to the formation of odd-numbered fatty acids. As a general consequence, heterotrophic fixation of CO2 should be taken into account in investigations of degradation experiments using isotope tracer compounds.