Two fluorescent calcium indicators, Fura 2 and Indo 1 were compared with Quin 2 for the measurement of cytosolic free calcium concentrations (Ca2+)1in intact platelets. The free calcium ions were quantitatively evaluated using a single excitation wavelength fluorimeter before and after stimulation with the Ca2+ionophores ionomycin and A23187, platelet activating factor (PAF) and arachidonic acid (AA). Using Quin 2 no rise in (Ca2+)icould be registered by stimulation with the weak agonists PAF and AA in subthreshold concentrations for aggregation in contrast to Indo 1 and, to a lesser extent, Fura 2. Stimulation with ionomycin and A23187 led to a significant rise in (Ca2+)idetected by all three indicators. The reproducibility was best for Indo 1. The three fluorescence indicators differed in their influence on platelet function and in their physical properties. Higher fluorescence intensities for Fura 2, and especially Indo 1, allowed a much lower indicator concentration of about 2–10% with a 10-fold improvement in the signal/noise ratio with Indo 1. While there was no difference in the volume distribution curves of the dye-loaded platelets, Quin 2-loaded platelets showed a significantly diminished response to collagen-and ADP-induced aggregation.