Extracts obtained from plants of Stevia rebaudiana Bert have high sweetening capacity and without risks for health. The sexual reproduction of this species is characterized by the low germination efficiency of its seeds and the high heterogeneity of the resulting populations. Thus, asexual propagation, particularly micropropagation, is the preferred tool for obtaining a large number of healthy and genetically homogeneous plants in a short time. To study the effects of the substrate composition and culture system on the nutrient contents of resulting plantlets, two stevia varieties (Criolla and Morita), three culture media (Murashige and Skoog and two modifications of this medium) and two culture systems (Temporal Immersion System-TIS-and solid medium) were used. After 30 days of culture the stem length and leaf nutritional status were tested. The nutrient contents of the in vitro well-developed plants are clearly related with the substrate compositions. Except for manganese no differences were observed between both varieties in terms of macro or micronutrients levels. In most cases the nutrient levels were higher in the microplants grown in solid medium. The macro and micronutrient levels of the resulting plantlets reported and discussed here, for the first time, may be useful for future studies in order to establish normal nutritional status of reference for researchers working in stevia micropropagation.