We describe a method for determining intracellular free calcium concentration ([Ca(2+)]) from single-wavelength fluorescence signals. In contrast to previous single-wavelength calibration methods, the proposed method does not require independent estimates of resting [Ca(2+)] but relies on the measurement of fluorescence close to indicator saturation during an experiment. Consequently, it is well suited to [Ca(2+)] 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 [Ca(2+)] 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 [Ca(2+)] was 32-59 nM in the proximal apical dendrite. Monitoring action potential-evoked [Ca(2+)] transients as a function of indicator loading yielded estimates of endogenous buffering capacity (44-80) and peak [Ca(2+)] changes at zero added buffer (178-312 nM). In young animals (postnatal days 14-17) our results were comparable to previous estimates obtained by ratiometric methods (, Biophys. J. 70:1069-1081), and no significant differences were seen in older animals (P24-28). We expect our method to be widely applicable to measurements of [Ca(2+)] and [Ca(2+)]-dependent processes in small neuronal compartments, particularly in the many situations that do not permit wavelength ratio imaging.