Soil microbial biomass C ( B c) is calculated from B c = F c k c in the original fumigation-incubation (FI) method. In this expression, k c is a proportionality constant to allow for the fact that not all the C in the microorganisms killed by the fumigant (CHCl 3) is evolved as CO 2 during the specified 10 day incubation. The flush ( F c) is taken as the CO 2 evolved in 10 days by the fumigated soil, less that evolved in 10 days by unfumigated soil — the ‘control’. Voroney and Paul (1984) argued that this unfumigated control should not be deducted, thus obtaining estimates of microbial biomass C that were much larger, often by a factor of 2 or more. At present some investigators use a control and some do not. This paper describes an experiment designed to resolve this issue. A grassland soil was incubated with uniformly 14C-labelled glucose for 27 days, then fumigated with CHCl 3. Labelled and unlabelled organic C extractable to 0.5 m K 2SO 4 were measured in the fumigated and unfumigated soils at this time and used to calculate E c, the amount of biomass C made extractable to K 2SO 4 by CHCl 3. Evolution of 14C-labelled and unlabelled CO 2 was also measured over the next 30 days in the fumigated and unfumigated soils. All the results were entirely consistent with the use of a control as proposed in the original method. Once the flush of decomposition had subsided, the CO 2 respiration curves of fumigated and unfumigated soil were nearly parallel. Following fumigation, the specific activity of the CO 2 evolved from the fumigated soil rapidly increased and then returned to a level close to that in the unfumigated soil, suggesting that the same pool of organic matter was being mineralised in both fumigated and unfumigated soils, once the heavily-labelled biomass killed by the fumigant had decomposed. Other evidence was that the specific activity of F c (139 Bq mg −1 C) did not differ significantly from that of E c (136 Bq mg −1 C), both calculated using the appropriate control.