Macrophages are critical mediators of disease throughout the course of HIV infection. They are one of the first cells to become infected and they act as significant viral reservoirs. HIV enters macrophages through binding to CD4 and CCR5 and the subsequent fusion events that occur are generally believed to be virus driven. However, evidence suggests that signaling through CCR5 and increases in intracellular calcium may facilitate HIV entry. Purinergic receptors, P2X and P2Y, are important mediators of the inflammatory response to pathogens and other causes of cellular damage. We hypothesize that HIV's binding to macrophages results in ATP release and autocrine activation of purinergic receptors, leading to increased intracellular calcium, which facilitates HIV replication. We examined HIV replication in primary human macrophages in the presence of oxidized ATP (oATP), a P2X receptor antagonist, and suramin, a P2X and P2Y antagonist. We found that both inhibitors significantly block viral replication by greater than 85%. Inhibition of replication by oATP is dose-dependent and results in a decrease in the percentage of cells infected. Using a viral entry assay, we also demonstrated that oATP inhibits HIV entry into macrophages. To determine what purinergic receptors are involved in viral replication, we used the more specific inhibitors NF 279 (P2X₁), A-740003 (P2X₇), and MRS 2179 (P2Y₁). All three antagonists significantly inhibited viral replication in a dose-dependent manner. Interestingly, only NF 279 inhibited viral entry, indicating that P2X₁ may be the predominant receptor mediating viral entry, while the other receptors participate in later stages of HIV replication. To determine how HIV's binding results in purinergic receptor activation, we examined ATP release following treatment with the HIV surface protein gp120. We found that gp120 induces a significant increase in ATP release from macrophages within the first 15 minutes of treatment. Our results identify a novel family of host proteins that participate in HIV replication, the P2 receptors, which may represent an appropriate target for therapeutic intervention.