Abstract A protocol for in vitro clonal propagation of a Brazilian variety of chamomile (Chamomilla recutita (L.) Rauschert) is described. Nodal segments were submersed in liquid MS basal medium containing 1.0–4.0 mg l−1 GA3 or thidiazuron (TDZ) or 0.5–4.0 mg l−1 2,4-D for 2 h. After that, they were transferred to solid MS basal media without growth regulators and maintained for 2 months. This process avoided the callus development which was undesirable for shoot multiplication. The best treatment for shoot proliferation and plant rooting contained 1.0–2.0 mg l−1 gibberellin (GA3) or 0.5 mg l−1 2,4-D. Plants treated with 0.5 mg l−1 2,4-D were subcultured for half-strength nitrogen solid MS medium and they were acclimatized two months later. The hardening process in the greenhouse was difficult and time consuming, however, 54% of plants survived and were successfully adapted to field conditions.