Orchids are known for the beauty, exuberance, and color of their flowers and therefore represent one of the most coveted ornamental plants. Due to their high commercial value, several illegal practices of removal and commercialization have led to the verge of extinction. This study aimed to develop protocols for in vitro conservation under slow growth and acclimatization of species of the subfamily Epidendroideae (Orchidaceae), from the state of Sergipe, Brazil. Two experiments were conducted: the first one tested four concentrations of MS (MURASHIGE and SKOOG, 1962) (25%, 50%, 75%, 100%), three orchid species (Catasetum macrocarpum, Oeceoclades maculata, and Polystachya estrellensis), and two temperatures (18 and 25ºC); the second one tested three combinations of carbon sources and osmotic regulators (20 g L -1 sucrose; 10 g L -1 sucrose + 5 g L -1 de mannitol; 10 g L -1 sucrose + 5 g L -1 sorbitol) and the same three species and two temperatures. For the in vitro conservation of C. macrocarpum, O. maculata, and P. estrellensis over 450 days, the use of 25% of MS salts or 20 g.L -1 sucrose at 25ºC is recommended. C. macrocarpum seedlings were acclimatized using sand + shredded pine bark + worm castings at a ratio of 2:2:1 (w:w:w). The use of sand alone is recommended for the acclimatization of O. maculata.