In vitro cultivation is a very important method for the conservation of germplasm and the multiplication of endangered plants; however, studies on the adequacy of the nutrients used for a good development of each species are needed. The objective of this study was to evaluate the in vitro development of Laelia marginata Lindl. in culture media containing different nitrate/ ammonium ratios. Seedlings were obtained from seeds germinated in vitro. The treatments consisted of different nitrate/ ammonium ratios, with five proportions of NO3 - and NH4 + : T1-0/100; T2-25/75; T3-50/50; T4-75/25 and T5-100/0. At 200 days after seedling transplanting, height of the aerial part, root length, number of leaves, number of shoots, leaf length, leaf width, aerial and root dry mass and contents of chlorophyll a, b and carotenoids were evaluated. A completely randomized design was used, with five treatments and ten replicates. The combination of 50/50 nitrate/ammonium resulted in the highest values of aerial part and root length, dry mass of the aerial part and root, as well as leaf length and width. The proportion of 50/50 nitrate/ammonium resulted in the best initial development of L. marginata seedlings.