Temperature fluctuations are a fundamental entity of the soil environment in the temperate zone and show fast (diurnal) and slow (seasonal) dynamics. Responses of soil respiration to temperature fluctuations were investigated in a root-free soil of a mid-European beech–oak forest. First, in laboratory we analysed the efflux of CO 2 from soil microcosms exposed to seasonal (±5 °C of the annual mean) and diurnal fluctuations (±5 °C of the seasonal levels) in a two-factorial design. Second, in field microcosms we investigated effects of smoothing diurnal temperature fluctuations in soil (simulating a possible global trend) on CO 2 efflux. Third, the natural temperature regime was simulated in laboratory microcosms and their CO 2 efflux was compared to the one in the field. The experiments lasted for 1 year to differentiate seasonal and annual responses. Dynamics of CO 2 efflux, microbial basal respiration, biomass and qO 2 varied with seasonal temperature regime. However, in the laboratory the annual cumulative CO 2-C production did not differ between treatments and varied between 10.9% and 11.7% of the total microcosm C, disregarding seasonal and/or diurnal fluctuations. The similarity of cumulative C production suggests that the availability of microbially mobilisable carbon pools rather than the temperature regime limited soil respiration. Diurnal fluctuations generally did not affect CO 2 efflux and microbial activity, though winter Q 10 values were increased in their absence. Simulation of the natural temperature regime in the laboratory resulted in CO 2 efflux similar to field microcosms. In the field, rates of CO 2 efflux and microbial activity, seasonal and annual cumulative CO 2-C production were significantly higher at smoothed than at natural temperature conditions (annually 13.1% and 11.0% of total C was respired, respectively). Facing global climate changes the mechanisms regulating responses of soil respiration to temperature fluctuations need further investigation.