Abstract: Objective To observe the effect of a new synthetic tripeptide [Arg(NO3)- Lys(OCH3)- Arg(NO3)] on L-arginine/NO pathway in the macrophage cell strain RAW246.7. Methods The cultured macrophages exposed to lipopolysaccharide (LPS, 1 μg/L) treatment were randomly divided into 3 groups (1=6) and treated with distilled water, 1×10-4 mol/L tripeptide and 1×10-4 mol/L L-arginine, NG-monomethyl-L-arginine (L-NMMA) for 24 h, respectively. The macrophages were incubated for 24 h with LPS (1 μg/L) and in the presence of different concentrations of L-arginine (0 to 2 mmol/L), or in normal culture medium (containing 0.5 mmol/L L-arginine) for 24 h with LPS (1 μg/L) and in the presence of tripeptide of 0 to 10×10-4 mol/L. The changes of [3H]-L-arginine transport and NO production from the macrophages were measured. Result NO release from macrophages incubated in the LPS-containing culture medium was 50 folds, and [3H]-L-arginine uptake 2.7 folds that in cells in normal culture medium. Tripeptide (1 ×10-4 mol/L) inhibited [3H]-L-arginine transport and NO production by 67% and 71% respectively. The effect of tripeptide was stronger than L-NMMA (P<0.05). Extracelluar L-arginine caused a concentration-dependent increase in nitrite production, which reached the maximum at concentrations above 0.5 mmol/L Km for nitrite production of 0.162±0.015 mmol/L andVmax of 91.2±2.3 μmol/(24h·106cells). L-arginine transport and NO production were inhibited by tripeptide, but their dose-dependent pattern of changes was different with EC50 of 0.21 ×10-4 mol/L and 1.27×10-4 mol/L, respectively. Conclusions Activation of macrophages with LPS induces nitrite accumulation in the culture medium, which is dependent on the presence of extracelluar L-arginine. The tripeptide induces dysfunction of L-arginine/NO pathway in macrophages, potently inhibits L-arginine transport and competitively combine the active sites of nitric oxide synthase.