Extracellular nucleotides can act as important intercellular signals in diverse biological processes, including the enhanced production of factors that are key to immune response regulation. One receptor that binds extracellular adenosine triphosphate released at sites of infection and injury is P2X(7), which is an ionotrophic receptor that can also lead to the formation of a non-specific pore, activate multiple mitogen-activated protein kinases (MAPKs), and stimulate the production of immune mediators including interleukin family members and reactive oxygen species (ROS). In the present report, we have investigated the signaling mechanisms by which P2X(7) promotes monocytic cell mediator production and induces transcription factor expression/phosphorylation, as well as how receptor-associated pore activity is regulated by intracellular trafficking. We report that P2X(7) stimulates ROS production in macrophages through the MAPKs ERK1/2 and the nicotinamide adenine dinucleotide phosphate oxidase complex, activates several transcription factors including cyclic-AMP response element-binding protein and components of the activating protein-1 complex, and contains specific sequences within its intracellular C-terminus that appear critical for its activity. Altogether, these data further implicate P2X(7) activation and signaling as a fundamental modulator of macrophage immune responses.