Extracellular ATP has been previously shown to activate calcium signalling in pituitary cell populations [1] but the particular cell types involved have not been identified. We used video imaging of Fura-2 loaded into single rat pituitary cells and identified as lactotrophs to study the effects of extracellular ATP on [Ca2+]i. ATP does not permeabilize the cells as shown by exclusion of propidium iodide. ATP causes two types of calcium transients in lactotrophs. The most common response is a rapid increase in [Ca2+]i that decays slowly and is terminated by washout of ATP. This type of response is also seen in calcium-free medium, demonstrating mobilization of calcium stores dependent upon the presence of the agonist. ATP also stimulates calcium entry as detected by Mn(2+)-quenching of Fura-2. ATP in Mg(2+)-free medium and ATP gamma S are effective agonists suggesting ATP4- is the active form. The presence of P2-purinoceptors is apparent because ATP, ADP and AMP increase [Ca2+]i in decreasing order of potency and adenosine has no effect. ATP-induced calcium transients are reduced by the P2-purinoceptor antagonists suramin and quinidine. UTP is equipotent with ATP and defines the receptor subtype as P2U. We conclude that ATP4- acts on rat lactotrophs via P2U-purinoceptors to elevate [Ca2+]i from intracellular and extracellular sources.