Our previous study revealed that proline (Pro) accumulated in peach trees when cold-acclimated shoots were exposed to higher temperatures. In this study, we determined whether such Pro accumulation is indeed a high temperature-stress response or metabolic preparation for growth resumption. Two studies were conducted to address this question using ‘Daewol’ peach trees. In study 1 (February), Pro content of eco-dormant shoots increased when cold-acclimated plants were exposed to two heat treatments (HTs) but remained unchanged during the intervening cold treatment (CT). Concurrently, ornithine-delta-aminotransferase (OAT) and Δ¹-pyrroline-5-carboxylate reductase (P5CR) genes were up- and down-regulated after two HTs and the CT, respectively. Study 2, conducted with growing trees (May), showed that Pro did not change and expression of P5CR and OAT was down-regulated when trees were subjected to either HT or CT; HT and CT used here constituted ‘stress’ treatments as revealed by the decline in chlorophyll fluorescence. Taken together, these results suggest that Pro accumulation (study 1) may be associated with the preparation for growth resumption and not ‘stress’. Furthermore, results from parallel gene expression studies suggest that the ornithine, rather than glutamate, pathway may be the primary Pro biosynthesis route both during growth resumption and high temperature stress in ‘Daewol’ peach.