Whole ecosystem carbon dioxide (CO2) exchange estimated with the eddy covariance (EC) technique has been central to studies on the responses of terrestrial ecosystems to disturbance and intra-annual and interannual variations in climate, but challenges exist in understanding and reducing the uncertainty in estimates of net ecosystem exchange (NEE) of CO2. We review the potential uncertainties associated with the eddy covariance technique, including systematic errors from insensitivity to high-frequency turbulence, random errors from inadequate sample size associated with averaging period, vertical and horizontal advection issues, and selection criteria for removing periods of inadequate mixing from further analyses. We also discuss benefits and caveats of using independent measurements to evaluate EC-derived NEE, such as comparisons of EC-derived annual NEE and allometric net ecosystem production estimates (NEP) and interpretation of nighttime NEE with scaled chamber-based estimates of ecosystem respiration.