Small explants composed of 3-6 layers of epidermal and subepidermal cells excised from floral branches of Nicotiana tabacum are capable of de novo organogenesis. Continuous mitoses without organogenesis, or mitoses followed by the formation of floral buds, vegetative buds or roots can be obtained by varying the auxin-cytokinin-sucrose ratio in the medium. The physiological stage of the floral branches, and light and darkness had also an influence on the de novo organogenesis. Best floral-bud formation was obtained when explants were taken from plants in which the terminal bud was in the green-fruit stage, and were cultured on a medium with indole-3-acetic acid and kinetin both at 10(-6) M and with 3% sucrose, in the light. For differentiation of vegetative buds, the optimal auxin/cytokinin ratio was one tenth of that for floral-bud formation, while the sucrose concentration was the same. Optimal conditions for root formation were: explants from plants in which the terminal bud carried a mature fruit; 10(-7) M kinetin, 10(-5) M indole-3-butyric acid, and 1% sucrose in the medium; and culture in darkness. The optimal medium for callus formation contained 5Γ10(-6)M 2,4-dichlorophenoxyacetic acid, 10(-7) M kinetin and 3% sucrose.