A new group of fluorescent indicators with visible excitation and emission wavelengths has been synthesized for measurements of cytosolic free Ca2+. The five compounds, "rhod-1," "rhod-2," "fluo-1," "fluo-2," and "fluo-3" (Figs. 2 and 3), combine the 8-coordinate tetracarboxylate chelating site of 1,2-bis(2-amino-phenoxyethane-N,N,N',N'-tetraacetic acid with a xanthene chromophore to give a rhodamine-like or fluorescein-like fluorophore. Binding of Ca2+ increases the fluorescence by up to 40-fold. The Ca2+ dissociation constants are in the range 0.37-2.3 microM, so that the new indicators should give better resolution of high [Ca2+] levels than previously obtainable with quin-2 or fura-2. The visible excitation wavelengths of the new compounds are more convenient for fluorescence microscopy and flow cytometry than the UV required by previous indicators. However, the new dyes' increase in fluorescence upon binding calcium is not accompanied by a wavelength shift, so they are unsuitable for measurements using ratios at two wavelengths. The most promising dye of this series is fluo-3, whose initial biological testing in fibroblasts is described in the following paper (Kao, J. P. Y., Harootunian, A. T., and Tsien, R. Y. (1989) J. Biol. Chem. 264, 8171-8178).