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We describe a method for determining intracellular free calcium concentration ([Ca²⁺]) from single-wavelength fluorescence signals. In contrast to previous single-wavelength calibration methods, the proposed method does not require independent estimates of resting [Ca²⁺] but relies on the measurement of fluorescence close to indicator saturation during an experiment. Consequently, it is well suited to [Ca²⁺] indicators for which saturation can be achieved under physiological conditions. In addition, the method requires that the indicators have large dynamic ranges. Popular indicators such as Calcium Green-1 or Fluo-3 fulfill these conditions. As a test of the method, we measured [Ca²⁺] in CA1 pyramidal neurons in rat hippocampal slices using Oregon Green BAPTA-1 and 2-photon laser scanning microscopy (BAPTA: 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid). Resting [Ca²⁺] was 32–59nM …