Biogenically released isoprene plays important roles in both tropospheric photochemistry and plant metabolism. We performed a 13CO 2-labeling study using proton-transfer-reaction mass spectrometry (PTR-MS) to examine the kinetics of recently assimilated photosynthate into isoprene emitted from poplar (Populus × canescens) trees grown and measured at different atmospheric CO 2 concentrations. This is the first study to explicitly consider the effects of altered atmospheric CO 2 concentration on carbon partitioning to isoprene biosynthesis. We studied changes in the proportion of labeled carbon as a function of time in two mass fragments, M41 +, which represents, in part, substrate derived from pyruvate, and M69 +, which represents the whole unlabeled isoprene molecule. We observed a trend of slower 13C incorporation into isoprene carbon derived from pyruvate, consistent with the previously hypothesized origin of chloroplastic pyruvate from cytosolic phosphenolpyruvate (PEP). Trees grown under sub-ambient CO 2 (190 ppmv) had rates of isoprene emission and rates of labeling of M41 + and M69 + that were nearly twice those observed in trees grown under elevated CO 2 (590 ppmv). However, they also demonstrated the lowest proportion of completely labeled isoprene molecules. These results suggest that under reduced atmospheric CO 2 availability, more carbon from stored/older carbon sources is involved in isoprene biosynthesis, and this carbon most likely enters the isoprene biosynthesis pathway through the pyruvate substrate. We offer direct evidence that extra-chloroplastic rather than chloroplastic carbon sources are mobilized to increase the availability of pyruvate required to up-regulate the isoprene biosynthesis pathway when trees are grown under sub-ambient CO 2.