Rhizome tips were the most suitable explants for in vitro plant regeneration and multiplication of Nymphaea hybrid ‘James Brydon’ on Murashige and Skoog medium containing different concentrations and combinations of indole-3-acetic acid, 1-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid, 6-benzyladenine (BA), kinetin, 2-isopentenyladenine (2iP) and gibberellic acid (GA3). A combination of 2iP, BA, and NAA strongly favored induction of shoot buds and shoot proliferation. Pretreatment of shoot cultures at 8°C for 30 days or with 14.4 or 28.9 μM GA3 for 15 days did not improve shoot multiplication. A 16-h photoperiod with photosynthetic photon flux of 30 μmol m-2 s-1 was found to be the optimum light condition for shoot growth and multiplication. Multiple shoots produced well developed root systems within 4 weeks after transfer to a plant growth regulator-free medium containing activated charcoal.