Isoprene is a biogenic hydrocarbon that significantly affects tropospheric chemistry. Numerous plant species, including many trees, emit isoprene. Isoprene is synthesized by isoprene synthase (IspS), from dimethylallyl diphosphate (DMADP) made by the methylerythritol 4-phosphate (MEP) pathway. It has been demonstrated that in developing leaves, isoprene emission is regulated by transcriptional control of IspS, while in mature leaves subjected to changing growth temperature, regulation of emission is shared between IspS and DMADP supply from the MEP pathway. Isoprene emission also varies throughout a day, with circadian regulation implicated. This study investigated changes in isoprene emission capacity, and expression of IspS and the enzymes of the MEP pathway throughout several days, with Populus trichocarpa grown at different temperatures to induce different levels of isoprene emission. Isoprene emission capacity exhibited ultradian regulation, with a period of about 12 h; peak capacity was observed at 0300 and 1500 h daily. Several of the enzymes of the MEP pathway had previously been suggested to have regulatory roles in the production of other plastidic terpenoids, and transcript accumulation for these enzymes, combined with in silico promoter analyses, supported a regulatory role for deoxyxylulose 5-phosphate synthase (DXS) in particular.