Serum glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) activity is reduced over 75% in systemic inflammatory response syndrome. To investigate the mechanism of this response, expression of the GPI-PLD gene was studied in the mouse monocyte-macrophage cell line RAW 264.7 stimulated with lipopolysaccharide (LPS; 0.5 to 50 ng/ml). GPI-PLD mRNA was reduced approximately 60% in a time- and dose-dependent manner. Oxidative stress induced by 0.5 mM H₂O₂ or 50 µM menadione also caused a greater than 50% reduction in GPI-PLD mRNA. The antioxidant N-acetyl-L-cysteine attenuated the down-regulatory effect of H₂O₂ but not of LPS. Cotreatment of the cells with actinomycin D inhibited down-regulation induced by either LPS or H₂O₂. The half-life of GPI-PLD mRNA was not affected by LPS, or decreased slightly with H₂O₂, indicating that the reduction in GPI-PLD mRNA is due primarily to transcriptional regulation. Stimulation with tumor necrosis factor alpha (TNF-) resulted in ~40% reduction in GPI-PLD mRNA in human A549 alveolar carcinoma cells but not RAW 264.7 cells, suggesting that alternative pathways could exist in different cell types for down-regulating GPI-PLD expression during an inflammatory response and the TNF- autocrine signaling mechanism alone is not sufficient to recapitulate the LPS-induced reduction of GPI-PLD in macrophages. Sublines of RAW 264.7 cells with reduced GPI-PLD expression exhibited increased cell sensitivity to LPS stimulation and membrane-anchored CD14 expression on the cell surface. Our data suggest that down-regulation of GPI-PLD could play an important role in the control of proinflammatory responses.