Effects of temperature and CO2 concentration on growth of in vitro plantlets (seedlings) of 'Amanatsu' (Citrus natsudaidai Hayata) were examined. Shoot length, number of leaves and roots of plantlets increased with increasing temperature in plant incubator. CO2 enrichment was effective for promoting growth of plantlets. Under 1,200 ppm CO2 condition, fresh weight both upper and underground parts of plantlets a month after transplant was significantly greater than that of the conventional culture condition without CO2 treatment. However, almost plantlets cultured under these conditions could be transplanted to pots without acclimatization, therefore, the significant differences among these treatments were not observed. Furthermore, the change with passage of time in CO2 concentration was estimated in tissue culture vessel containing a plantlet. In the closed vessel, the CO2 was perfectly consumed by photosynthesis of the plantlet under the light condition. While, in the culture vessel with a disc of microporous polypropylene film under 1,200 ppm CO2 condition, the CO2 was consumed to the concentration level of approximately 700 ppm. In the both treatments under the dark period, the CO2 was accumulated to the maximum concentration of approximately 2,000 ppm.