The interaction between plant hormones throughout plant regeneration and development is complex. In the present study, we extended our investigation and disclosed the capacity of de novo shoot organogenesis from cotyledon explants of bottle gourd in relation to plant hormones and ethylene inhibitor. Synergistic effect of kinetin and benzyl adenine (BA) enhanced the shoot regeneration efficiency (80.6%) from cotyledon explants of bottle gourd, compared with BA or kinetin used separately, even in the presence of AgNO₃. The use of BA or kinetin separately or in synergistic combination led to a characteristic brownish color of the explants at the bud formation stage which was related to ethylene production. In the cotyledon explants cultured on MS medium supplemented with kinetin, the production of ethylene was lower than when BA or the combination of BA and kinetin was used. However, the regeneration capacity was very low and the emergence of shoot buds was delayed. BA promoted ethylene production while the synergistic effect of kinetin and BA decreased ethylene production and played a role in high frequency of regeneration from cotyledon explants of bottle gourd. The physiological relevance of the hormonal interaction in relation to the regulation of ethylene action and effect on regeneration was discussed.