Leptocarpha rivularis, commonly known as "palo negro", is a species of the Astereaceae family, endemic in the forests of Southern Chile. The extracts of this plant have medicinal properties attributable to its secondary metabolites, mainly identified as flavonoids and terpenes. Among them, leptocarpine has gained importance as a powerful anticarcinogen. The aim of this study was thein vitroestablishment ofL. rivularisto develop future alternatives to obtain biomass for medicinal purposes.Young shoots obtained from plants in growth chambers were treated with Captan (TM) (2.5% (w/v)), subjected to disinfection method with ethanol, commercial chlorine, and antioxidants and tested in different Murashige & Skoog based media to establishin vitroculture. The most effective growth was observed with half strength Murashige & Skoog medium supplemented with 20 g L(-1)sucrose and 3.2 g L(-1)gelrite (MGS medium) with 95% survival at 30-d and shooting reaching 82% of the explants when Plant Preservative Mixture (TM) is added to the medium. The addition of 1 mg L(-1)of indole-3-butyric acid to the MSG medium was necessary for inducing roots and obtaining well-rooted plants that were acclimatized and successfully established in the greenhouse. Our study represents the first report describing thein vitroculture ofL. rivularis, allowing the sustainable use of this plant.