Microglial cells are the major cellular elements with immune function inside the CNS and play important roles in orchestrating inflammatory brain response to hypoxia and trauma. Although a complete knowledge of the endogenous factors leading to a prompt activation of microglia is not yet available, activation of P2 purinoreceptors by extracellular ATP has been indicated as a primary factor in microglial response. A still unresolved question, however, is which subtype(s) of P2 receptors mediate(s) the response to ATP. By a combination of RT-PCR, Western blotting, and single-cell calcium imaging, we assessed the presence and the activity of P2 receptor subtypes in the mouse microglial cell line N9. All members of the P2 receptor family, including the recently reported receptor for sugar nucleotides (P2Y(14)), were found to be present in these cells at mRNA and/or protein level. The functionality of the receptors was assessed by analysis of the calcium responses evoked by specific agonists both in N9 cells and in primary microglia from rat brain. Interestingly, a different functional profile of P2 receptors was observed in resting or in LPS-activated N9 cells. Overnight exposure to LPS increased P2Y(6) and P2Y(14), decreased P2X(7), and left unchanged P2Y(1) and P2Y(2,4) receptor activity. The change in the P2 receptor profile in activated cells suggests selective roles for specific P2 receptor subtypes in microglial activation triggered by LPS. We speculate that modulation of microglial cell function via subtype-selective P2 receptor ligands may open up new strategies in the therapeutic management of inflammatory neurological diseases characterized by abnormal microglia response.