1. Modulation of intracellular free Ca2+ concentration ([Ca2+]i) by extracellular ATP was investigated in cultured adult rat brown adipocytes using the fluorescent Ca2+ indicator fura-2. 2. Bath application of ATP in micromolar concentrations caused a large increase in [Ca2+]i in cells previously stimulated with noradrenaline. This ATP-induced [Ca2+]i increase exhibited a monotonic decline to near the resting levels within approximately 2 min, even in the continued presence of the agonist. 3. The magnitude and time course of the [Ca2+]i increase in response to ATP were not significantly affected by removal of extracellular Ca2+, suggesting that a mobilization of intracellular Ca2+ primarily contributes to the increase. 4. The [Ca2+]i increase in response to ATP was sensitive to inhibition by suramin, suggesting the involvement of P2 purinoceptors in the response. 5. Thapsigargin (100 nM) evoked a gradual and irreversible increase in [Ca2+]i which was entirely dependent upon extracellular Ca2+, providing functional evidence for the expression of store-operated Ca2+ entry in these brown adipocytes. 6. Extracellular ATP at a concentration of 10 microM depressed this thapsigargin (100 nM)-induced [Ca2+]i increase by 92 +/- 3 % (n = 8 cells), strongly suggesting that ATP inhibits an influx of Ca2+ across the plasma membrane through the store-operated pathway. Bath application of phorbol 12-myristate 13-acetate (PMA, 5 microM) did not affect the thapsigargin-induced [Ca2+]i increase, indicating that the inhibitory action of ATP is not mediated by activation of protein kinase C (PKC). 7. These results indicate that extracellular ATP not only mobilizes Ca2+ from the intracellular stores but also exerts a potent inhibitory effect on the store-operated Ca2+ entry process in adult rat brown adipocytes.