The carbon-isotopic composition (delta13C) of soil-respired CO2 has been employed to evaluate soil carbon-cycling processes and the contribution of soil CO2 emissions to canopy and tropospheric air. These evaluations can be successful only when accurate isotope values of soil-respired CO2 are available. Here, we tested the robustness of delta13C values of soil-respired CO2 obtained after long incubations in static closed chambers that were initially flushed with soil air. The rationale of this approach is that the equilibrium carbon-isotope values of chamber-headspace CO2 are theoretically equal to those of CO2 produced within the soil. Static closed chambers were installed in replicated grass monocultures, and measurements of headspace CO2 concentrations and delta13C values were performed at regular time intervals for 24 h in July 2005. The results revealed no significant effects of grass species on headspace CO2 concentrations or delta13C values (repeated measures analysis of variance (ANOVA), P>0.1). As predicted by theory, isotope values asymptotically approached equilibrium conditions, which in our experimental setting occurred after 10 h. This good match between model predictions and our results suggests that an accurate determination of delta13C values of CO2 produced within soils is obtained through the isotopic measurement of chamber-headspace CO2 once equilibrium conditions have been reached with the underlying soils. An additional advantage of this approach is that only one sample per chamber is required, which, combined with the low uncertainties of these measurements, facilitates the investigation of the spatial (landscape) variability of soil-respired CO2.