The activity of GA 20-oxidase (GA20ox), a 2-oxoglutarate-dependent dioxygenase, is a critical regulatory factor in the gibberellin-(GA)-biosynthetic pathway. Two genes, HaGA20ox1 and HaGA20ox2, along with their corresponding cDNAs exhibiting structural features and homology to GA 20-oxidases of several plant species, have been isolated from vegetative shoots of sunflower (Helianthus annuus). Sequence analysis revealed that both genes consist of three exons and two introns. GA20ox genes from dicot and monocot species showed similar structures. The amino acid sequences deduced from the sunflower cDNA clones showed high level of similarity, with identities of HaGA20ox1/HaGA20ox2 of 84%. The phylogenetic analysis indicated that all monocot and all dicot GA20ox were grouped in two separate clusters. Within the dicot clade, HaGA20ox1 and HaGA20ox2 formed a subclade with GA 20-oxidases from Lactuca sativa (Ls20ox1 and Ls20ox2), Chrysanthemum x morifolium (DgGA20ox1), and Daucus carota (DcGA20ox2). Both HaGA20ox1 and HaGA20ox2 shared the highest similarity with the L. sativa Ls20ox1. The high accumulation of both HaGA20ox transcripts coincided with the period of rapid growth of the sunflower embryo, suggesting a role for GA in the first phase of embryo maturation. HaGA20ox1 mRNA was also detected in all the organs tested but occurred at a higher level in the vegetative shoot and the root, whereas HaGA20ox2 transcripts were preferentially accumulated in inflorescence meristems, vegetative shoots, internodal stem, and roots. Treatment of sunflower plants with an inhibitor of GA biosynthesis showed that HaGA20ox2, but not HaGA20ox1, was subject to feedback regulation by a reduction of bioactive GAs.