Isoprene basal emission (the emission of isoprene from leaves exposed to a light intensity of 1000 µmol m−2 s−1 and maintained at a temperature of 30 °C) was measured in Phragmites australis plants growing under elevated CO2 in the Bossoleto CO2 spring at Rapolano Terme, Italy, and under ambient CO2 at a nearby control site. Gas exchange and biochemical measurements were concurrently taken. Isoprene emission was lower in the plants growing at elevated CO2 than in those growing at ambient CO2. Isoprene emission and isoprene synthase activity (IsoS) were very low in plants growing at the bottom of the spring under very rich CO2 and increased at increasing distance from the spring (and decreasing CO2 concentration). Distance from the spring did not significantly affect photosynthesis making it therefore unlikely that there is carbon limitation to isoprene formation. The isoprene emission rate was very quickly reduced after rapid switches from elevated to ambient CO2 in the gas-exchange cuvette, whereas it increased when switching from ambient to elevated CO2. The rapidity of the response may be consistent with post-translational modifications of enzymes in the biosynthetic pathway of isoprene formation. Reduction of IsoS activity is interpreted as a long-term response. Basal emission of isoprene was not constant over the day but showed a diurnal course opposite to photosynthesis, with a peak during the hottest hours of the day, independent of stomatal conductance and probably dependent on external air temperature or temporary reduction of CO2 concentration. The present experiments show that basal emission rate of isoprene is likely to be reduced under future elevated CO2 levels and allow improvement in the modelling of future isoprene emission rates.