Soil is a net source of CO₂, because roots and microorganisms are mineralizing plant residues and soil organic matter. However, also the reverse process, namely CO₂ fixation, occurs and may have significant implications for the assessment of bound residue formation as determined by the use of radiolabelled pollutants as well as for the isotopic composition of microbial biomass - if exposed to atmospheric CO₂ - and soil organic matter. We incubated an arable soil under an atmosphere enriched with ¹³CO₂ in the dark for up to 81 days to study the processes involved in CO₂ fixation by soil microorganisms and the fate of the CO₂-derived carbon in soil. CO₂ was fixed along with heterotrophic microbial activity in the soil. The amount of CO₂ fixed corresponded to about 7% of the microbial biomass and was linearly correlated with respiration. The process was almost completely suppressed by fumigation with CHCl₃. We found ¹³C-enrichment in the fatty acids and the amino acids, but not in the amino sugars. This points to maintenance metabolism of the soil microbial cells without significant growth during the incubation. For each individual fatty acid, more label was found in the total fraction (microbial biomass + non-living organic matter) than in the polar lipid fatty acid fraction (microbial biomass only). Part of the label thus already has been transferred from the biomass to non-living soil organic matter. The amino acids carrying the highest label were asparagine and aspartate, indicating oxaloacetate as a direct product of CO₂ fixation. The reaction therefore is related to anaplerotic reactions in heterotrophic organisms. Our results show that CO₂-fixation by heterotrophic microorganisms is an essential process of the carbon cycle in soil and thus also contributes to the formation of soil organic matter.