Thyroid cells express a variety of both P2Y and P2X purinergic receptor subtypes. G-protein coupled P2Y receptors are known to influence a wide variety of thyrocyte specific functions, however, functional P2X receptor gated channels have not been observed. In this study, we used whole cell-patch clamp recording and fluorescence imaging of the plasma membrane marker FM1-43 to examine the effects of extracellular ATP on membrane permeability and trafficking in the Fisher rat thyroid cell line, FRTL. We found a cationic selective current that was gated by ATP and 2',3'-O-(4-benzoylbenzoyl)-ATP (Bz-ATP), but not by UTP. The ATP evoked currents were inhibited by pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid (PPADS), adenosine 5'-triphosphate-2'3'-dialdehyde (oxo-ATP), 100 µM Zn²⁺, and 50 µM Cu²⁺. Fluorescence imaging revealed pronounced, temperature-sensitive, stimulation of exocytosis and membrane internalization by ATP with the same pharmacological profile as observed for activation of current. The EC₅₀ for ATP stimulation of internalization was 440 µM in saline containing 2 mM Ca²⁺ and 2 mM Mg²⁺, and 33 µM in low Mg²⁺, nominally Ca²⁺-free saline. Overall, the results are most consistent with activation of a P2X₇ receptor by ATP^4-. However, low permeability to N-methyl-D-glucamine⁺+ (NMDG⁺) and the propidium cation YO-PRO-1 indicates absence of the cytolytic pore that often accompanies P2X₇ receptor activation. ATP stimulation of internalization occurs in Na⁺-free, Ca²⁺-free, or low Mg²⁺ saline, and therefore, does not depend on cation influx through the ATP-gated channel. It is concluded that ATP activation of a P2X₇ receptor stimulates membrane internalization in FRTL cells via a transduction pathway that does not depend on cation influx.