The nitrate reductase (NR)-defective double mutant of Arabidopsis thaliana (nia1 nia2) has previously been shown to present a low endogenous content of NO in its leaves compared with the wild-type plants. In the present study, we analyzed the effect of NR mutation on floral induction and development of A. thaliana, as NO was recently described as one of the signals involved in the flowering process. The NO fluorescent probes diaminofluorescein-2 diacetate (DAF-2DA) and 1,2-diaminoanthraquinone (1,2-DAA) were used to localize NO production in situ by fluorescence microscopy in the floral structures of A. thaliana during floral development. Data were validated by incubating the intact tissues with DAF-2 and quantifying the DAF-2 triazole by fluorescence spectrometry. The results showed that NO is synthesized in specific cells and tissues in the floral structure and its production increases with floral development until anthesis. In the gynoecium, NO synthesis occurs only in differentiated stigmatic papillae of the floral bud, and, in the stamen, only anthers that are producing pollen grains synthesize NO. Sepals and petals do not show NO production. NR-deficient plants emitted less NO, although they showed the same pattern of NO emission in their floral organs. This mutant blossomed precociously when compared with wild-type plants, as measured by the increased caulinar/rosette leaf number and the decrease in the number of days to bolting and anthesis, and this phenotype seems to result from the markedly reduced NO levels in roots and leaves during vegetative growth. Overall, the results reveal a role for NR in the flowering process. © The Author 2008. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. 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