In a system of endotoxin (LPS)-mediated NO production in ANA-1 murine macrophages, suppression subtractive hybridization was used to identify genes up-regulated by NO. Osteopontin (OPN), a secreted acidic phosphoprotein that binds to a cell surface RGD integrin-binding motif, was found to be differentially expressed in the presence of NO. OPN has been demonstrated to inhibit NO production in a variety of cell types. Northern blot and nuclear run-on analyses demonstrated that OPN mRNA levels and gene transcription were significantly increased in the presence of LPS-induced NO synthesis. Transient transfection of an OPN promoter-luciferase reporter plasmid construct showed that promoter activity is increased in the presence of LPS and NO. Immunoblot analysis showed that OPN protein is secreted into the extracellular fluid. Similar results were noted with an alternative cell system, RAW 264.7 macrophages, and alternative inducers of NO synthesis, IFN-γ and IL-1β. In the presence of GRGDSP, a hexapeptide that blocks binding of RGD-containing proteins to cell surface integrins, NO production is significantly increased in the presence of LPS stimulation. These data suggest a unique trans-regulatory mechanism in which LPS-induced NO synthesis feedback regulates itself through up-regulation of OPN promoter activity and gene transcription.