To further our understanding of the influence of global climate change on isoprene production we studied the effect of elevated [CO2] and vapour pressure deficit (VPD) on isoprene emission rates from leaves of Populus deltoides Bartr. during drought stress. Trees, grown inside three large bays with atmospheres containing 430, 800, or 1200 μmol mol-1 CO2 at the Biosphere 2 facility, were subjected to a period of drought during which VPD was manipulated, switching between low VPD (approximately 1 kPa) and high VPD (approximately 3 kPa) for several days. When trees were not water-stressed, elevated [CO2] inhibited isoprene emission and stimulated photosynthesis. Isoprene emission was less responsive to drought than photosynthesis. As water-stress increased, the inhibition of isoprene emission disappeared, probably as a result of stomatal closure and the resulting decreases in intercellular [CO2] (Ci). This assumption was supported by increased isoprene emission under high VPD. Drought and high VPD dramatically increased the proportion of assimilated carbon lost as isoprene. When measured at the same [CO2], leaves from trees grown at ambient [CO2] always had higher isoprene emission rates than the leaves of trees grown at elevated [CO2], demonstrating that CO2 inhibition is a long-term effect.