The aim of the present study was to investigate the relationship between agonist‐induced changes in intracellular free Ca²⁺ ([Ca²⁺]_j) and the refilling of intracellular Ca²⁺ stores in Fura 2–loaded thyroid FRTL‐5 cells. Stimulating the cells with ATP induced a dose‐dependent increase in ([Ca²⁺]_j). The ATP‐induced increase in [Ca²⁺]_j was dependent on both release of sequestered intracellular Ca²⁺ as well as influx of extracellular Ca²⁺. Addition of Ni²⁺ prior to ATP blunted the component of the ATP‐induced increase in [Ca²⁺]_j dependent on influx of Ca²⁺. In cells stimulated with ATP in a Ca²⁺‐free buffer, readdition of Ca²⁺ induced a rapid increase in [Ca²⁺]_j; this increase was inhibited by Ni²⁺. In addition, the ATP‐induced influx of ⁴⁵Ca²⁺ was blocked by Ni²⁺. Stimulating the cells with noradrenaline (NA) also induced release of sequestered Ca²⁺ and an influx of extracellular Ca²⁺. When cells were stimulated first with NA, a subsequent addition of ATP induced a blunted increase in [Ca²⁺]_j. If the action of NA was terminated by addition of prazosin, and ATP was then added, the increase in [Ca²⁺]_j was restored to control levels. Addition of Ni²⁺ prior to prazosin inhibited the restoration of the ATP response. In the presence of extracellular Mn²⁺, ATP stimulated quenching of Fura 2 fluorescence. The quenching was probably due to influx of Mn²⁺, as it was blocked by Ni²⁺. The results thus suggested that stimulating release of sequestered Ca²⁺ in FRTL‐5 cells was followed by influx of extracellular Ca²⁺ and rapid refilling of intracellular Ca²⁺ stores.