Using cotyledonary explants excised from seedlings germinated in vitro, efficient plant regeneration via organogenesis was established for two winter squash (Cucurbita maxima Duch.) cultivars. To establish optimal conditions for adventitious shoot induction, a variety of explants were prepared from seedlings of different ages and these were cultured using media containing different concentrations of 6-benzylaminopurine (BA). For both cultivars. plant regeneration was optimal when the proximal parts of cotyledons from 4-day-old seedlings were cultured on induction medium composed of Murashige and Skoog (MS) medium with 1 mg/l BA. After 3 weeks of culture in induction medium, 82 and 92% of explants from the two cultivars regenerated shoots. Adventitious shoots were subcultured on elongation medium composed of MS medium with 0.1 mg/l BA and the elongated shoots were successfully rooted in MS medium without growth regulators for 2 weeks. Flow cytometric analysis revealed that most of the regenerated plants were diploid. (C) 2003 Elsevier Science Ireland Ltd. All rights reserved.