Heat-induced rosetted Eustoma grandiflorum requires low temperature for induction of stem elongation and flowering. Although heat-induced rosetting is associated with a reduction of gibberellin A1 (GA1) content, how thermo-induction affects GA biosynthesis is unclear. Thus, we examined levels of GA, precursors including that of ent-kaurene which is the first committed step in GA biosynthesis. We used uniconazole, an ent-kaurene oxidase inhibitor to estimate the ent-kaurene biosynthesis activity.The accumulation level of ent-kaurene in stems of the cold-treated seedlings was approximately 1.8 times that of the non-cold-treated seedlings, whereas no difference was observed in the leaves. No change was observed in endogenous levels of GA1 and GA20 in stems of the heat-induced rosetted plants during the cold treatment, whereas their levels increased with stem elongation after transfer to warm conditions. In contrast to the levels of GA1 and GA20, endogenous levels of ent-kaurene, ent-kaurenoic acid, GA53, GA44 and GA19 in the stems markedly increased at the end of cold treatment. These results indicate that ent-kaurene biosynthesis and its metabolism early in the GA biosynthetic pathway are stimulated by low temperature and, later, the stimulation leads to an increment of endogenous levels of GA1 which is essential for stem elongation of the heat-induced rosetted E. grandiflorum