Underground adventitious buds of leafy spurge undergo three well-defined phases of dormancy, para-, endo-, and ecodormancy, throughout the year. A hormone that participates in the regulation of various plant developmental processes including dormancy and growth is ethylene. In this study, we examined ethylene involvement during growth of leafy spurge buds, particularly after paradormancy release (decapitation). Our results did not show an obvious increase in ethylene during bud growth. However, when buds were incubated with 1 mM ACC, ethylene levels were higher in growing than non-growing buds, suggesting that the levels of the ethylene biosynthetic enzyme ACC oxidase increased in growing buds. Real-time qPCR indicated that the transcript of an Euphorbia esula ACC oxidase (Ee-ACO) increased up to 4-fold after growth induction following decapitation. Immunoblot analyses identified a 36-kD Ee-ACO protein that increased in expression during bud growth. In addition, Ee-ACO is highly expressed in leaves, moderately expressed in crown buds, stems and meristems, and weakly expressed in roots and flowers. Immunolocalization on sections of growing buds revealed strong labeling of Ee-ACO in the nucleus and cytoplasm of young cells at the shoot apical meristem and leaf primordia. An exception to this pattern occurred, however, in cells undergoing mitosis, where labeling of Ee-ACO was negligible. These observations suggest that ethylene is synthesized in young cells of growing buds, but only during certain stages of the cell cycle.