In vitro culture of mycotrophic leaf-bearing seedlings of federally threatened Platanthera praeclara Sheviak and Bowles, a terrestrial orchid native to the midwestern prairies, is reported for the first time. Symbiotic germination was evaluated to: (1) determine need for cold moist stratification to enhance seed germination and seedling development and (2) identify the mycobionts that support in vitro germination and development. Germination was improved by exposing seeds to both 4- and 6-mo stratification periods; whereas seeds without stratification failed to germinate in a pilot study. Pretreatment of seeds with 6 mo of stratification combined with inoculation with mycorrhizal fungus derived from a seedling (Ceratorhiza sp., UAMH 9847) supported development of higher stage protocorms and some leaf-bearing seedlings of P. praeclara. Protocorms with developing leaf primordia were also obtained by coinoculation with strains of Epulorhiza and Ceratorhiza. Fungi derived from mature P. praeclara plants failed to promote seedling development to advanced growth stages. Results indicate that in vitro development of P. praeclara is best supported when stratified seeds are cultured with fungi isolated from young seedlings.