The effect of soil aeration status on carbon partitioning of a labelled organic substrate (14C-[U]-glucose) into CO2, microbial biomass, and extra-cellular metabolites is described. The soil was incubated in a continuous flow incubation apparatus under four different aeration conditions: (1) permanently aerobic, (2) permanently anaerobic, (3) shifted from anaerobic to aerobic, and (4) shifted from aerobic to anaerobic. The soil was pre-incubated for 10 days either under aerobic or under anaerobic conditions. Afterwards, glucose was added (315 μg C g−1) and the soils were incubated for 72 h according to four treatments: aerobic or anaerobic conditions maintained, aerobic conditions shifted to anaerobic conditions and anaerobic conditions shifted to aerobic conditions. Carbon partitioning was measured 0, 8, 16, 24, 48 and 72 h after the glucose addition. In permanently aerobic conditions, the largest part of the consumed glucose was built into microbial biomass (72%), much less was mineralised to CO2 (27%), and only a negligible portion was transformed to soluble extra-cellular metabolites. Microbial metabolism was strongly inhibited when aeration conditions were changed from aerobic to anaerobic, with only about 35% of the added glucose consumed during the incubation. The consumed glucose was transformed proportionally to microbial biomass and CO2. In permanently anaerobic conditions, 42% of the consumed glucose was transformed into microbial biomass, 30% to CO2, and 28% to extra-cellular metabolites. After a shift of anaerobic to aerobic conditions, microbial metabolism was not suppressed and the consumed glucose was transformed mainly to microbial biomass (75%) and CO2 (23%). Concomitant mineralisation of soil organic carbon was always lower in anaerobic than in aerobic conditions.