To measure the concentrations of cytosolic ionized calcium in platelets we used a calcium-sensitive fluorophor (Fura-2) with two different spectrofluorometers (Perkin-Elmer LS-5 and Fluorolog 222). Values obtained by these two instruments for the basal cytosolic ionized calcium concentration of resting platelets and those of agonist-activated platelets did not differ significantly. Both instruments were capable of monitoring the shifts in wavelengths induced by the dye-calcium complex, the ratio between absorbances at the two wavelengths (340/380 nm), and calcium concentrations continuously during agonist-induced platelet activation. We conclude that a relatively inexpensive instrument may be adequate for measuring ionized calcium in cells by this method, although sophisticated kinetic studies may require analytical or research-grade instruments.