Cannabis sativa L. (Cannabaceae) is a multi-use plant, valued all over the world yielding fiber and food, as well as a psychoactive drug. In vitro regeneration of Cannabis provides one approach for mass clonal propagation of this important crop. Shoots were proliferated from nodal segments and were maintained over a period of more than 2 years by sequential subculture on a medium containing 0.5µmol l–1TDZ . De novo regeneration was induced on nodal segments cultured onto a medium containing thidiazuron. Axenic cultures were also used to test the three different cultivation systems (temporary immersion bioreactor, synthetic seed technology and semi solid culture conditions) for shoot multiplication of C. sativa. Growth of plantlets in a temporary immersion bioreactor resulted in significant increases in biomass, shoot multiplication rate, shoot height and transplant efficiency. Plantlets grown in the bioreactors were rooted and acclimatized under growroom conditions. Together, these experiments have established optimized parameters for propagation and growth of C. sativa plantlets in a sterile controlled environment for biochemical characterization and production of high-quality medicinal products. Acknowledgment: This work was supported with federal funds from the National Institute of Drug Abuse (NIDA), National Institute of Health (NIH), Department of Health and Human Services, USA, under the contract No. N01DA-7–7746. References:  Lata H, Chandra S, et al. (2009) In vitro Cellular and Developmental Biol: Plant, 45(1): 12–19.