Unregulated increasing of Tumor necrosis factor-alpha (TNF-alpha) could be pathogenic in inflammatory diseases. The aim of this study was to investigate the anti-inflammatory role of the Substance P-antagonists (SPAs) through the inhibition of histamine release (HR) and TNF-alpha production from mast cell. Rat peritoneal mast cells (PMC) stimulated with Substance P (SP), in the presence of SPAs or not, were analyzed for HR and TNF-alpha protein production. Competitive Polymerase Chain Reaction, with an internal standard competing with target cDNA for the same primers, was used to determine the TNF-alpha mRNA expression. We show that the increase of either HR and TNF-alpha levels in peritoneal (PMC) after induction with SP was inhibited by pre-incubation with SPA or with the Peptide 101 (P101), while the [D-Pro2, D-Phe7, D-Trp9]-SP (dSP) had no effect. Neuraminidase treatment suggests that dSP, as well as SP, interacts with sialic acid residues on the cell surface. Moreover, SPA and P101 also inhibit the release of histamine and TNF-alpha induced by dSP suggesting that a receptor-independent mechanism is involved. These data could be useful to better understand the mechanisms involved in the mast cell activation and TNF-alpha production in the inflammatory diseases where SP is involved.