Down-regulation of cytokines is implicated as an important component of the phenomenon of tolerance to bacterial products in humans and animals. Since many effects of endotoxin and cytokines are mediated by nitric oxide, this study was designed to evaluate in vivo the L-arginine:NO pathway in endotoxin tolerance. Sublethal injections of E. coli lipopolysaccharide (LPS, 1 mg/kg body wt, i.p.) were given to rats daily for five days. Blood levels of NO2-/NO3-, stable metabolites of nitric oxide (NO), significantly increased on day 1 (baseline, 89.64 ± 40, day 1, 260.32 ± 36 nmol/ml; P < 0.05) but returned to baseline levels on day 5 (77.60 ± 5 nmol/ml). However, urinary NO2-/NO3- remained significantly elevated several-fold throughout the study period (baseline, 121.25 ± 11.4, day 1, 899.35 ± 15.8, day 5, 250.23 ± 21.4 nmol/hr/100 g body wt). Glomeruli and aortae obtained from these rats showed increased NO production that was maintained at similar levels even at day 5 (glomeruli: control, 0.01 ± 0.0, day 1, 22.4 ± 0.3, and day 5, 22.0 ± 2.5, P < 0.05 vs. control; aortae: control, 0.01 ± 0.0, day 1, 4.3 ± 2.2, and day 5, 5.4 ± 1.0 nmol/hr/mg protein, P < 0.05 vs. control, respectively); this further increased significantly in response to in vitro LPS challenge. However, peritoneal macrophages, liver and spleen showed an initial increase in NO production that decreased significantly by the fifth day of LPS and could not be further stimulated by in vitro LPS challenge. Thus, in vivo NO synthesis is down-regulated during protracted LPS. Our results show that the process is relatively specific to the liver, spleen and macrophages, and is qualitatively and quantitatively different in vascular tissues such as aortae and glomeruli.