Native scrub-oak communities in Florida were exposed for three seasons in open top chambers to present atmospheric [CO2] (approx. 350 μmol mol−1) and to high [CO2] (increased by 350 μmol mol−1). Stomatal and photosynthetic acclimation to high [CO2] of the dominant species Quercus myrtifolia was examined by leaf gas exchange of excised shoots. Stomatal conductance (gs) was approximately 40% lower in the high- compared to low-[CO2]-grown plants when measured at their respective growth concentrations. Reciprocal measurements of gs in both high- and low-[CO2]-grown plants showed that there was negative acclimation in the high-[CO2]-grown plants (9–16% reduction in gs when measured at 700 μmol mol−1), but these were small compared to those for net CO2 assimilation rate (A, 21–36%). Stomatal acclimation was more clearly evident in the curve of stomatal response to intercellular [CO2] (ci) which showed a reduction in stomatal sensitivity at low ci in the high-[CO2]-grown plants. Stomatal density showed no change in response to growth in high growth [CO2]. Long-term stomatal and photosynthetic acclimation to growth in high [CO2] did not markedly change the 2·5- to 3-fold increase in gas-exchange-derived water use efficiency caused by high [CO2].