An efficient regeneration system was established for an ethnomedicinal shrub Rhinacanthus nasutus from root-derived callus organogenesis. The root segments were cultured on MS medium supplemented with various concentrations of Kn (1.0-4.0 mu M) alone or in combination with IBA (0.2-0.6 mu M) or 2, 4-D (0.5-1.5 mu M). The optimum frequency (94 %) of callus induction was recorded on MS medium supplemented with 3.0 mu M Kn and 0.4 mu M IBA. For shoot regeneration from callus, MS medium supplemented with different concentrations (1.0-7.0 mu M) of BA or TDZ alone or in combination with NAA (0.2-1.0 mu m) was employed. The highest frequency of shoot regeneration (91 %) and mean number of shoots (28.3) were observed on MS medium supplemented with 5.0 mu M BA and 0.7 mu M NAA. The shoots were excised and cultured on MS medium with 4.0 mu M IBA produced 3.4 roots per shoot in 88 % cultures. Of the 65 plants transferred to soil 54 survived (83 %). The plants were transferred to field after successful hardening. RAPD analysis of the regenerated plants showed high similarity with the mother plant.