Neutrophils play a critical role in early immunity to many microbial pathogens, and this may in part be due to their ability to release immunoregulatory cytokines and chemokines during infection. Here, we demonstrate by flow cytometric analysis that mouse polymorphonuclear leukocytes (PMN) up-regulate surface expression of TNF-alpha within 10 min of stimulation with LPS, and that this is followed by gradual loss over a period of 18 h. Early increases in surface TNF-alpha expression correlated with loss of intracellular pools of preformed TNF-alpha. Nevertheless, extended incubation with LPS resulted in increased levels of TNF-alpha mRNA synthesis and replenishment of intracellular cytokine. After triggering with LPS, PMN acquired the ability to induce dendritic cell (DC) TNF-alpha and IL-12 production. Transwell assays demonstrated that high-level DC TNF-alpha production induced by LPS-triggered neutrophils was dependent upon cell-to-cell contact and neutrophil TNF-alpha, but neither was required for neutrophil instruction of DC IL-12 synthesis. The data suggest that microbial Ag-triggered mouse PMN acquire the capacity to deliver potent DC-activating signals through elaboration of cytokines and direct interactions at the cell surface.