The object of this work was to study the ability of methane oxidizing bacteria to use CO2 as an acceptor of electrons liberated in methane oxidation and the role of CO2 fixation in the constructive metabolism of the bacteria. All of the studied methane oxidizing bacterial cultures were found to be capable of fixing the 14C of hydrocarbonate. The activity of the process was shown to be similar in different strains. Up to 30% of the carbon in the biomass composition could originate from the carbon of HCO3-. Methane oxidizing bacteria that assimilated C1-compounds via the hexulose phosphate and serine pathways had the same level of HCO3- fixation. No differences were found among strains of the same species, among species, or among genera. The assimilation of HCO3- was catalyzed by PEP-carboxylase (i. e. in a heterotrophous way) or, in some cultures, by ribulose-1,5-diphosphate carboxylase, the key enzyme in the autotrophous pathway of CO2 assimilation. The enzymological mechanisms of HCO3- assimilation are discussed. The biological role of CO2 fixation in the metabolism of methane oxidizing bacteria that use the hexulose phosphate and serine pathways of methane assimilation may be different. The process can either play the role of anapleurotic reactions in the tricarboxylic acid cycle, or be an element of the serine pathway of methane assimilation. Calculations have shown that the extent to which a substrate to be metabolized is reduced seems to determine the activity of exogenous CO2 fixation. The contribution made by HCO3- fixation into the carbon metabolism of methane oxidizing bacteria confirms that they are related to lithotrophous organisms.