Previous work with spinach (Spinacia oleracea) has shown that the level of gibberellin (GA) 20-oxidase is strongly up-regulated by long days (LD). In the present work, the effect of photoperiod on expression of other GA dioxygenases was investigated and compared with that of GA 20-oxidase. Two GA 2-oxidases and one GA 3-oxidase were isolated from spinach by reverse transcription-polymerase chain reaction with degenerate primers and by 5'- and 3'-rapid amplification of cDNA ends. As determined by high-performance liquid chromatography with on-line radioactivity detection, the SoGA3ox1 gene product catalyzed 3beta-hydroxylation of GA(9) to GA(4) and GA(20) to GA(1). The SoGA2ox1 and the SoGA2ox2 gene products catalyzed 2beta-hydroxylation of GA(9) to GA(51) and GA(20) to GA(29). The product of GA(20) metabolism by SoGA3ox1 was identified as GA(1) by gas chromatography-mass spectrometry, whereas the products of GA(1) and GA(20) metabolism by SoGA2ox1 and SoGA2ox2 were identified as GA(8) and GA(29), respectively. SoGA2ox1 also metabolized GA(53) to GA(97). The levels of SoGA20ox1 transcripts were greatly increased in all organs tested in LD conditions, but the levels of SoGA3ox1 transcripts were only slightly increased in blades and petioles. A decrease in the levels of the SoGA2ox1 transcripts in young leaves and tips in LD conditions is opposite to the expression pattern of the SoGA20ox1. Expression of SoGA20ox1 in petioles and young leaves was strongly up-regulated by a supplementary 16 h of light, but the levels of SoGA3ox1 and SoGA2ox1 transcripts did not change. It is concluded that regulation and maintenance of GA(1) concentration in spinach are primarily attributable to changes in expression of SoGA20ox1.