To help unravel the role of brassinosteroids (BRs) in the control of shooting, we treated the shoots of Marubakaido apple rootstock (Malus prunifolia (Willd.) Borkh cv. Marubakaido) with brassinolide and Brz 220, an inhibitor of BR biosynthesis. Brassinolide differentially affected elongation and formation of main and primary lateral shoots, which resulted in reduced apical dominance. Treatment of shoots with increasing doses of Brz 220 led to a progressive inhibition of main shoot elongation. Eight different BRs were also identified in the shoots of M. prunifolia. Progressive decline in 6-deoxocathasterone, 6-deoxotyphasterol and castasterone was related to increased doses of Brz 220. Analysis of the metabolic profiles between a fluoro-containing derivative of 28-homocastasterone (5F-HCS) using treated and untreated shoots demonstrated that no 5F-HCS-specific metabolite was identified. However, 4 weeks after the treatment, fructose, glucose and the putatively identified gulonic acid were higher in 5F-HCS-treated shoots, compared to untreated shoots. These results indicate that the previously reported 5F-HCS-induced stimulation of shoot elongation and formation of new shoots in the Marubakaido shoots is under the control of changes in the endogenous BR pool. In addition, the results presented in this report also indicate that the 5F-HCS-induced shooting likely involves a variety of different mechanisms and consequently does not result from changes in the endogenous levels of any single metabolite.