The nonallelic gib-1 and gib-3 tomato (Lycopersion esculentum Mill.) mutants are gibberellin deficient and exhibit a dwarfed growth habit. Previous work has shown that this dwarfed growth pattern can be reversed by the application of a number of gibberellins and their precursors, including ent-kaurene (ent-kaur-16-ene). This indicates that they are blocked in gibberellin biosynthesis at a step prior to ent-kaurene metabolism. The normal accumulation of carotenoids observed in these mutants suggests a functionally normal isoprenoid pathway. Ent-kaurene is synthesized from geranylgeranyl pyrophosphate in a two-step process with copalyl pyrophosphate as an intermediate. In vitro assays using young fruit extracts from wild-type and gib-2 plants resulted in the conversion of geranylgeranyl pyrophosphate to copalyl pyrophosphate, and the conversion of copalyl pyrophosphate to entkaurene. Similar assays using gib-1 plants indicated a reduced ability for synthesis of copalyl pyrophosphate from geranylgeranyl pyrophosphate, and thus a reduced ent-kaurene synthetase A activity. Furthermore, gib-3 extracts demonstrated a reduced ability to synthesize ent-kaurene from copalyl pyrophosphate, and thus a reduced ent-kaurene synthetase B activity. These results establish the enzymatic conversion of geranylgeranyl pyrophosphate to copalyl pyrophosphate, and copalyl pyrophosphate to ent-kaurene, as the sites of the mutations in gib-1 and gib-3 tomatoes, respectively. We also note that tomato fruit extracts contain components which are inhibitory to ent-kaurene synthesis. © 1990 Springer-Verlag New York Inc.