Soil CO2 efflux is a major component of net ecosystem productivity (NEP) of forest systems. Combining data from multiple researchers for larger-scale modeling and assessment will only be valid if their methodologies provide directly comparable results. We conducted a series of laboratory and field tests to assess the presence and magnitude of soil CO2 efflux measurement system × environment interactions. Laboratory comparisons were made with a dynamic, steady-state CO2 flux generation apparatus, wherein gas diffusion drove flux without creating pressure differentials through three artificial soil media of varying air-filled porosity. Under these conditions, two closed systems (Li-6400-09 and SRC-1) exhibited errors that were dependent on physical properties of the artificial media. The open system (ACES) underestimated CO2 flux. However, unlike the two other systems, the ACES results could be corrected with a single calibration equation that was unaffected by physical differences in artificial media. Both scale and rank changes occurred among the measurement systems across four sites. Our work clearly shows that soil CO2 efflux measurement system × environment interactions do occur and can substantially impact estimates of soil CO2 efflux. Until reliable calibration techniques are developed and applied, such interactions make direct comparison of published rates, and C budgets estimated using such rates, difficult.