Traditional breeding and molecular approaches have been used to develop tobacco varieties with reduced nicotine and secondary alkaloid levels. However, low-alkaloid (LA) tobacco varieties often show impaired leaf ripening and senescence leading to poor leaf quality upon curing. An analysis of the nicotine biosynthesis pathway suggests possible interactions with polyamine and ethylene pathways, potentially having an influence on leaf ripening and senescence. Therefore, we investigated the role of polyamines in tobacco leaf ripening by analyzing free, soluble conjugated and insoluble conjugated polyamine fractions (putrescine, spermidine and spermine) in leaves and roots of LA and normal alkaloid (NA) tobacco varieties grown in the field and a greenhouse. Data revealed that levels of free and conjugated forms of putrescine and spermidine were higher in LA plants compared to NA controls. Fractions of the conjugated forms increased with leaf ripening and senescence in roots and leaves of LA plants. Furthermore, a positive correlation between polyamines, chlorophyll content and phenotype development in LA plants was observed. Treatments of LA plants with polyamine biosynthesis inhibitors reduced free putrescine content in roots and leaves similar to NA plant levels, thus leading to partial reversion of the LA phenotype; however, conjugated polyamines and chlorophyll levels were not affected. Our data indicate that the unbalanced crosstalk among nicotine, polyamine and ethylene biosynthesis is involved in delayed senescence but may not be the only factor responsible for impaired leaf ripening.