Gibberellins (GAs) are essential for the development of fertile flowers in tomato, and may also be required immediately after fertilization. In the GA-biosynthetic pathway, the reactions catalyzed by GA 20-oxidases have been implicated as site of regulation. To study the regulation of GA biosynthesis in flower and early fruit development, we isolated three tomato GA 20-oxidase cDNA clones, Le20ox-1, -2 and -3. The three genes showed different organ-specific patterns of mRNA accumulation. Analysis of the transcript levels of the three GA 20-oxidase genes, as well as those of copalyl diphosphate synthase (LeCPS) and GA 3 beta-hydroxylase (Le3OH-2) during flower bud and early fruit development, revealed temporally distinct patterns of mRNA accumulation. Up until anthesis, transcripts were observed for LeCPS, Le20ox-1, -2 and Le3OH-2, with an accumulation of Le20ox-1 mRNA. In contrast to the high level of Le3OH-2 transcripts in the fully open flower, mRNA levels of Le20ox-1, -2 and LeCPS were reduced at this stage. After anthesis, LeCPS and Le20ox-1 transcripts increased again. In addition, Le20ox-3transcripts increased whereas the transcripts of Le3OH-2 decreased to an undetectable level. In situ hybridization results demonstrated that during early stages of bud development, Le20ox-2 transcripts were localized in the tapetum and placenta. The presented results supply novel data about localization of GA biosynthesis gene transcripts, and indicate that transcript levels of GA biosynthesis genes are all highly regulated during flower bud development.