Simultaneous measurements of net ecosystem CO2 exchange (NEE) were made in a Florida scrub-oak ecosystem in August 1997 and then every month between April 2000 to July 2001, using open top chambers (NEEO) and eddy covariance (NEEE). This study provided a cross validation of these two different techniques for measuring NEE. Unique characteristics of the comparison were that the measurements were made simultaneously, in the same stand, with large replicated chambers enclosing a representative portion of the ecosystem (75 m2, compared to approximately 1–2 ha measured by the eddy covariance system). The value of the comparison was greatest at night, when the microclimate was minimally affected by the chambers. For six of the 12 measurement periods, night NEEO was not significantly different to night NEEE, and for the other periods the maximum difference was 1.1 µmol m−2s−1, with an average of 0.72 ± 0.09 µmol m−2s−1. The comparison was more difficult during the photoperiod, because of differences between the microclimate inside and outside the chambers. During the photoperiod, air temperature (Tair) and air vapour pressure deficits (VPD) became progressively higher inside the chambers until mid-afternoon. In the morning NEEO was higher than NEEE by about 26%, consistent with increased temperature inside the chambers. Over the mid-day period and the afternoon, NEEO was 8% higher that NEEE, regardless of the large differences in microclimate. This study demonstrates both the uses and difficulties associated with attempting to cross validate NEE measurements made in chambers and using eddy covariance. The exercise was most useful at night when the chamber had a minimal effect on microclimate, and when the measurement of NEE is most difficult.