The influence of sucrose concentration (10 and 30 g l-1), low level of nitrogen salts (1/8 KNO3, 1/8 NH3NO4), auxins (IBA 1 mg l-1 + IAA 1 mg l-1 + NAA 0,01 mg l-1), and temperature (15?C, 20?C) on the rooting in vitro and acclimatization in the greenhouse of two cultivars ('Jadwiga', 'Professor Wojcicki') of Paeonia lactiflora were investigated. The level of endogenous carbohydrates in the peony shoots and roots during the rooting and acclimatization phases were analysed. There were a higher rooting and acclimatization percentage of 'Professor Wojcicki' cultivar than 'Jadwiga' cultivar . Also, more roots were produced by shoots of 'Professor Wojcicki' cultivar . The presence of auxins in the medium and the higher concentration of sucrose (30 g l-1) stimulated number of roots/shoot. On the other hand, the higher rooting percentage was found on the auxin?free medium in the presence of high level of sucrose. The shoots of 'Professor Wojcicki' rooted best when cultured at 20?C, but the shoots of 'Jadwiga' at lower temperature - 15?C. During the rooting stage, the major sugars detected in the peony microplants were sucrose, glucose and fructose. After four weeks of acclimatization, the plantlets accumulated starch and showed strong inhibition of shoot growth, and dormant buds were developed.