Arabidopsis thaliana leaves were infiltrated with DanePy (3-(N-diethylaminoethyl)-N-dansyl)aminomethyl-2,5-dihydro-2,2,5,5-tetramethyl-1H-pyrrole), a double, fluorescent and spin sensor of singlet oxygen. DanePy fluorescence was imaged by laser scanning microscopy. We found that DanePy penetrated into chloroplasts but did not alter the functioning of the photosynthetic electron transport as assessed by chlorophyll fluorescence induction. In imaging, DanePy fluorescence was well distinct from chlorophyll fluorescence. Photoinhibition by excess photosynthetically active radiation caused quenching of DanePy fluorescence in the chloroplasts but not in other cell compartments. When leaves were infiltrated with dansyl, the fluorescent group in DanePy, there was no fluorescence quenching during photoinhibition. This shows that the fluorescence quenching of DanePy is caused by the conversion of its pyrrol group into nitroxide, i.e. it was caused by the reaction of singlet oxygen with the double sensor and not by artifacts. These data provide direct experimental evidence for the localization of singlet oxygen production to chloroplasts in vivo.