We have developed an accurate and practical method for measuring intracellular Ca2+ concentration [( Ca2+]i) in single cells in monolayer culture using the fluorescent Ca2+-binding dye quin2. Quin2 was loaded into cells as a membrane-permeant ester which is hydrolyzed in the cytoplasm to the impermeant free acid, which is the indicator form (Tsien, R.Y., T. Pozzan, and T.J. Rink, 1982, J. Cell Biol., 94:325-334). The method involves the measurement of fluorescence at 340-nm excitation (I340), where dye fluorescence is dependent on Ca2+, and at 360-nm excitation (I360), where dye fluorescence is independent of Ca2+. The ratio of these two values (I340/I360) is thus related to the concentration of Ca2+ but independent of dye concentration and can be used as a measure of [Ca2+]. To test the ratio method in the microscope, we measured [Ca2+]i in GH3 cells in monolayer culture. We found a resting [Ca2+]i of 44 +/- 28 nM (mean +/- SD, n = 34), as compared with a suspension value (Gershengorn, M., and C. Thaw, 1983, Endocrinology, 113:1522-1524) of 118 +/- 18 nM. We also measured [Ca2+]i during stimulation of the cells with thyrotropin-releasing hormone (TRH) and found a 2.4-fold increase above resting levels within 20 s, a trough at 73% of resting at 90-100 s, and a peak slightly above resting at 3 min. Depolarization of the plasma membrane with KCl produced a sustained increase in [Ca2+]i. All of these data are in good agreement with the results of Gershengorn and Thaw on suspension cultures. When measuring both resting [Ca2+]i and the effects of TRH and KCl on small groups of cells, we found some variation among experiments. Using an image intensifier-video camera, we videotaped cells during TRH stimulation. Digital image analysis of these pictures demonstrated that there was a large variation in responsiveness from cell to cell. The microscope ratio method offers the possibility of resolving regions of differing [Ca2+] within the cytoplasm.