ATP-gated P2X(4) receptors (P2X(4)R) in macrophages and microglia have been implicated in neuropathic and inflammatory pain by currently unidentified mechanisms. P2X(4)R are found predominantly in intracellular lysosomal compartments but can be rapidly trafficked to the surface membrane by procedures that induce endolysosomal secretion. We studied total and surface membrane P2X(4)R protein expression by Western blot and biotinylation assays and functional expression by whole-cell patch clamp assays in human and rat alveolar macrophages in response to phagocytosis of zymosan and opsonized zymosan bioparticles and to classical and alternative macrophage activation. Unstimulated macrophages showed high total protein expression but very low functional expression. Phagocytosis rapidly (within 4 h) increased functional P2X(4)R expression by 2- to 7-fold as did chloroquine, an agent known to induce lysosomal secretion. In contrast, classical activation of macrophage for 48 h with IFN-gamma and TNF-alpha or IFN-gamma and LPS reduced surface and functional P2X(4)R expression by 3-fold without altering total P2X(4)R protein levels. Alternative activation with IL-4 or IL-13 did not alter total, surface or functional expression of P2X(4)R. This is the first study of the regulation of P2X(4)R in macrophages by physiological stimuli and presents a picture whereby P2X(4)R become functional in response to initial phagocytic stimuli but return to a non-functional state during sustained activation by classical macrophage activation.