4',9'-Bis(diphenylphosphinyl)naphthofluorescein (PNF-1) has been designed and synthesized as a highly selective, sensitive, cell-permeable, red fluorescent probe for detecting O(2) (.-) in biological systems. The design strategy for the probe is based on the nucleophilic mechanism of O(2) (.-) to mediate deprotection of the probe to naphthofluorescein, the emission spectrum of which is just in the spectral region of low background fluorescence interference in biological systems. Upon treatment with O(2) (.-), the probe exhibits a strong fluorescence response and high selectivity for O(2) (.-), rather than other reactive oxygen species or biological compounds. A linear calibration curve for PNF-1 showed a detection limit of 0.1 nM O(2) (.-). This new type of fluorescent probe allows nanomolar changes in O(2) (.-) concentrations in living cells to be detected by confocal microscopy.