Effects of photoautotrophic and photomixotrophic growth conditions on adventitious shoot regeneration from leaf explants of eastern cottonwood (Populus deltoides Bartr. ex Marsh.) were investigated. Rooting and proliferating shoot cultures (Stage I) were grown in either an elevated (1500 ppm) CO2 concentration (CO2) at high photosynthetic photon flux (PPF; approximately 150 μmol m⁻² s⁻¹) (photoautotrophic condition) with 0, 10 or 30 g l⁻¹ sucrose or under standard conditions (ambient (360 ppm) CO2 at low PPF (approximately 60 μmol m⁻² s⁻¹) with 30 g l⁻¹ sucrose). Leaves harvested from these cultures were analyzed for soluble sugars and were used as explants for adventitious shoot regeneration (Stage II), which was also carried out under photoautotrophic and standard conditions. Photoautotrophic conditions during Stage I promoted growth of rooting shoots but inhibited axillary shoot proliferation. Photoautotrophic conditions during Stage II suppressed callus and adventitious bud production from leaf explants compared with standard conditions. The regeneration environment appeared to be more important in controlling bud formation than the conditions under which the donor shoots were grown. Regardless of Stage I treatment, bud production was up to 100-fold higher for leaves cultured under standard conditions than under photoautotrophic conditions. Once adventitious buds were differentiated from the leaf tissues, however, their elongation was faster under photoautotrophic conditions than that under standard conditions, with some shoots reaching 10 mm in length on leaf explants cultured under photoautotrophic conditions. Because total leaf soluble sugar concentration was always lowest in shoots under standard conditions, which also yielded the highest bud production, the results suggest that endogenous starvation enhanced shoot production.