Purinergic G protein-coupled receptors (P2YR) and ion-conducting receptor-channels (P2XR) are present in the pituitary. However, their identification, expression within pituitary cell subpopulations, and the ability to elevate intracellular Ca2+ concentration ([Ca2+]i) in response to ATP stimulation were incompletely characterized. Here we show that mixed populations of rat anterior pituitary cells express messenger RNA transcripts for P2Y2R, P2X2aR, P2X2bR, P2X3R, P2X4R, and P2X7R. The transcripts and functional P2Y2R were identified in lactotrophs and GH3 cells, but not in somatotrophs and gonadotrophs, and their activation by ATP led to an extracellular Ca2+-independent rise in [Ca2+]i in about 40% of cells tested. Lactotrophs and GH3 cells, but not somatotrophs, also express transcripts for P2X7R, P2X3R, and P2X4R. Functional P2X7R were identified in 74% of lactotrophs, whereas 50% of these cells expressed P2X3R and 33% expressed P2X4R. Coexpression of these receptor subtypes in single lactotrophs was frequently observed. Purified somatotrophs expressed transcripts for P2X2aR and P2X2bR, and functional receptors were identified in somatotrophs and gonadotrophs, but not in lactotrophs. Consistent with the cell-specific expression of transcripts for P2X2R and P2X3R, the expression of their functional heteromers was not evident in pituitary cells. Receptors differed in their capacities to elevate and sustain Ca2+ influx-dependent rise in [Ca2+]i during the prolonged ATP stimulation. These results indicate that the purinergic system of anterior pituitary is extremely complex and provides an effective mechanism for generating a cell- and receptor-specific Ca2+ signaling pattern in response to a common agonist.