The radical nitric oxide (NO) is an important signaling molecule in plants and modulates several metabolic and developmental processes, including flowering. The transition from vegetative to reproductive growth is delayed in mutant plants that overproduce NO whereas flowering is precocious in mutant plants deficient in NO synthesis (Crawford, 2006, J. Exp. Bot. 57: 471-478). NO can be synthesized in plants during the oxidation of L-arginine to L-citrulline by a nitric oxide synthase (NOS) that has no homology with animal NOS. In addition to L-arginine, nitrite is an important source of NO in plants, and this pathway of NO generation depends on nitrate reductase (NR) activity, which reduces nitrate to nitrite during nitrogen assimilation. The aim of the present study was to identify the sources and sites of NO synthesis during flower development. We used the fluorescent NO indicator diaminofluorescein diacetate and epifluorescence and confocal microscopy to localize and quantify the sites of NO production in Arabidopsis thaliana flowers in different developmental stages. We also compared the synthesis of NO in the floral structures of wild type plants with that of mutants deficient in NOS (nos1) or NR (nia1 nia2) genes. NO was synthesized in specific cells and tissues of the floral structure and its production increased during floral development until anthesis. In the gynoecium, NO synthesis occurred only in differentiated stigmatic papillae of the floral bud. This synthesis was unaltered in NR-deficient plants, but was reduced in NOS-deficient plants, indicating that in these cells NO was derived mainly from L-arginine. In the stamen, only anthers that were producing pollen grains synthesized NO, and this production was independent of NR. In the stamen filament, there was no NO production in NR-deficient plants whereas NO biosynthesis occurred in Atnos1 mutant and wild-type plants, indicating that NO synthesis in the filament derived from nitrite. Sepals and petals showed no significant NO production in the three genotypes analyzed. These results indicate that NO is produced from different substrates and at specific sites and stages of flowering. The controlled production of NO during flower development strengthens the idea that this radical is an important signaling molecule in plant reproduction.