Valeriana glechomifolia is an endemic species of southern Brazil, capable of accumulating, in all of its organs, the terpene derivatives known as valepotriates, the presumed sedative components of the roots of pharmaceutically used species of Valeriana. In vitro cultures of the plant were established and the accumulation of acevaltrate, didrovaltrate, and valtrate in callus, cell suspension, and untransformed root cultures was studied. Leaves of in natura plants and roots of micropropagated plantlets were used as the explants for callus induction and root culture establishment, respectively, on Gamborg B5 basal medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) alone or with kinetin (KIN). Culture growth and secondary metabolite yields were enhanced with 2,4-D (4.52 μM) and KIN (0.93 μM). Maximum valepotriate contents, quantified by HPLC, of acevaltrate (ACE) 2.6 mg g⁻¹ DW, valtrate (VAL) 10.2 mg g⁻¹ DW, and didrovaltrate (DID) 2.9 mg g⁻¹ DW were observed in root cultures after 7–8 wk of culture.