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The cellular and subcellular localization of endogenous nitric oxide (NO ^˙) in leaves from young and senescent pea (Pisum sativum) plants was studied. Confocal laser scanning microscopy analysis of pea leaf sections with the fluorescent probe 4,5-diaminofluorescein diacetate revealed that endogenous NO ^˙ was mainly present in vascular tissues (xylem and phloem). Green fluorescence spots were also detected in the epidermal cells, palisade and spongy mesophyll cells, and guard cells. In senescent leaves, NO ^˙ generation was clearly reduced in the vascular tissues. At the subcellular level, by electron paramagnetic resonance spectroscopy with the spin trap Fe(MGD)₂ and fluorometric analysis with 4,5-diaminofluorescein diacetate, NO ^˙ was found to be an endogenous metabolite of peroxisomes. The characteristic three-line electron paramagnetic resonance spectrum of NO ^˙, with g = 2.05 and a_N = 12 …