Deuterium- and tritium-labeled gibberellins (GAs) were applied to stems of 3-year-old Eucalyptus globulus Labill. saplings and 9-month-old potted seedlings. Cambial region tissues surrounding the application point were collected 6, 24 or 48 h later. Twenty-four hours after application of 5 &mgr;g of [(2)H(2)]GA(20), 7% of the cambial region GA(20) pool, 7% of the GA(1) pool and 58% of the GA(29) pool were labeled with deuterium based on selected ion monitoring of purified extracts subjected to gas chromatography-mass spectrometry. The relatively low percent dilution of endogenous GAs by [(2)H(2)]GAs suggests that the exogenous application of [(2)H(2)]GA(20) did not result in substrate overloading, indicating that these conversions probably occur naturally within cambial region tissues. Extracts from similar cambial region tissues fed tritium-labeled GAs were sequentially fractionated by SiO(2) partition chromatography, C(18) reversed phase HPLC and N(CH(3))(2) HPLC. The radioactivity profiles indicated metabolism of GA(20) to GA(1) and GA(29), GA(1) conversion to GA(8), GA(4) to GA(34) and GA(9) to GA(51). Gibberellins GA(34), GA(51) and GA(29) are C-2beta-hydroxylated catabolites of low biological activity, whereas GA(1) and GA(4) are probably effectors of growth in the Eucalyptus stem and shoot. Evidence for C-13 hydroxylation of GA(4) to GA(1), GA(9) to GA(4) or GA(9) to GA(20) in the stem was inconclusive. Thus, although GA(4) and GA(9) are native to cambial region tissues, GA(1) is probably not produced from them in significant quantities. We conclude that the early C-13-hydroxylation pathway; i.e., conversion of GA(19) to GA(20) to GA(1), is the major pathway of GA(1) biosynthesis.