Biogenic isoprene emission has widespread impacts on air quality and tropospheric chemistry.  Rising atmospheric CO₂ is predicted to indirectly increase isoprene emission rates through stimulated plant productivity, but may also have a direct effect on isoprene synthesis.  We tested the general theory that isoprene-emitting forests will exhibit reduced isoprene emissions in the future through a direct biochemical response to higher CO₂ concentrations.  We conducted numerous experiments using chamber-grown poplar and aspen as well as aspen and sweetgum growing in Free Air CO₂ Enhancement (FACE) experiments.   In all cases, we observed a significant reduction in the isoprene emission rate per unit leaf area when trees were grown at elevated CO₂.  This suppression of the isoprene emission rate may be the result of an upregulation in expression of the phosphoenolpyruvate carboxylase (PEPc) gene, which shifts patterns of cytosolic and chloroplastic substrate use and limits the availability of pyruvate substrate for chloroplastic isoprene biosynthesis.  The magnitude of this direct reduction of isoprene emission was higher than observed increases in photosynthesis rate or net primary productivity in all cases, indicating that it is likely to offset any increases in isoprene emission rate due to increased leaf biomass.  Thus, the direct effect of CO₂ on isoprene emissions must be accounted for in accurate models of future emission responses.