Leaf isoprene emission scales positively with light intensity, is inhibited by high carbon dioxide (CO 2 ) concentrations, and may be enhanced or inhibited by low oxygen (O 2 ) concentrations, but the mechanisms of environmental regulation of isoprene emission are still not fully understood. Emission controls by isoprene synthase, availability of carbon intermediates, or energetic cofactors have been suggested previously. In this study, we asked whether the short-term (tens of minutes) environmental control of isoprene synthesis results from alterations in the immediate isoprene precursor dimethylallyldiphosphate (DMADP) pool size, and to what extent DMADP concentrations are affected by the supply of carbon and energetic metabolites. A novel in vivo method based on postillumination isoprene release was employed to measure the pool size of DMADP simultaneously with the rates of isoprene emission and net assimilation at different light intensities and CO 2 and O 2 concentrations. Both net assimilation and isoprene emission rates increased hyperbolically with light intensity. The photosynthetic response to CO 2 concentration was also hyperbolic, while the CO 2 response curve of isoprene emission exhibited a maximum at close to CO 2 compensation point. Low O 2 positively affected both net assimilation and isoprene emission. In all cases, the variation in isoprene emission was matched with changes in DMADP pool size. The results of these experiments suggest that DMADP pool size controls the response of isoprene emission to light intensity and to CO 2 and O 2 concentrations and that the pool size is determined by the level of energetic metabolites generated in photosynthesis.