Nodal explants of tomato (Lycopersicon esculentum Mill.) were cultured in vitro to evaluate the effects of sugar concentration, photosynthetic photon flux (PPF), CO2 concentration, ventilation rate of the vessel, and leaf removal on growth and photosynthesis. After 20 days of culture, the dry weights of plantlets derived from explants with leaves and cultured photoautotrophically (without sugar in the medium) under high PPF, high CO2 concentration, and high ventilation rate were more than twice as great as those of plantlets derived conventionally from explants without leaves and cultured photomixotrophically (with sugar in the medium) under low PPF, low CO2 concentration, and low ventilation rate (107 and 45 mg per plantlet, respectively). Under photomixotrophic micropropagation conditions, the dry weights of plantlets from explants with leaves increased more than did those of plantlets from explants without leaves. High PPF, high CO2 concentration, and high ventilation rate increased net photosynthetic rate and promoted growth of the plantlets under photomixotrophic micropropagation conditions. Photomixotrophic conditions produced the greatest dry weight and the longest shoots, but photoautotrophic conditions produced the highest net photosynthetic rate. The number of leaves did not differ significantly between photoautotrophically and photomixotrophically cultured plantlets. Thus, photoautotrophic micropropagation is applicable to the production of high quality tomato transplants.