Spilanthes oleracea Jacq. has been extensively used due to its pharmacological activities, and it has been considered as a promising vegetable crop. Nodal-segment cultures of S. oleracea were established in order to analyze the content of alkamide spilanthol. On average, 11 buds/nodal segment were developed after 30 days on MS basal salts liquid medium containing 2.22 μM benzyladenine (BA). Regenerated shoots formed complete plantlets in medium without growth regulators, and 80.2% of the regenerated plantlets survived in field conditions. Higher production of spilanthol (58.5% relative area) was obtained from the leaves and stems of plants grown in liquid medium up to 90 days, than in fieldgrown plants. Addition of BA to the culture medium significantly affected spilanthol accumulation. The production of spilanthol was also detected on the shoot basal callus. The spilanthol content varied with the plant organ in micropropagated plants. Ethanolic extract of leaves from field-grown plants showed no acute toxicology in Swiss mice at a dosage of 3 g/kg.