The involvement of nitric oxide (NO) in tolerance development to endotoxin has been proposed because peripherally administered NG-nitro-L-arginine methyl ester (L-NAME) (NO synthases inhibitor) delays the endotoxin tolerance formation. Since L-NAME is capable of crossing the blood-brain barrier, the question arises of where activity of NO synthases (inside or outside the blood-brain barrier) is crucial for development of endotoxin tolerance. To clarify the role of different NO synthases (NOS) isoforms, acting in the brain, on the tolerance development, effects of highly selective iNOS and nNOS inhibitors on stepwise attenuation of febrile response during tolerance formation were examined in freely moving biotelemetered rats. We monitored changes in febrile response during the development of tolerance to repeated intraperitoneal (i.p.) injections of lipopolysaccharide (LPS) (50 μg/kg) along with intracerebroventricular (i.c.v.) injections of vinyl-L-NIO, a neuronal NOS inhibitor, or aminoguanidine, an inducible NOS inhibitor at a dose of 10 μg/rat. Both inhibitors injected at the selected doses had no effect on normal day-time as well as night-time body temperature. Rats were treated with LPS and NOS inhibitors for three consecutive days. On the fourth day, all rats were injected with LPS alone. Rats repeatedly injected with LPS became tolerant to pyrogenic effect of LPS as early as on the second day of the experiment. The treatment with iNOS or nNOS inhibitors completely suppressed fever due to the first, second and third LPS injection. When rats, which received the three i.c.v. injections of vL-NIO along with i.p. injections of LPS, were then treated the fourth time with LPS alone, they responded with virtually identical changes in body temperature to that of the group of rats that were injected with water i.c.v. and LPS i.p. for three consecutive days. This data indicate that both group of rats became tolerant to pyrogenic effect of LPS. It is, therefore, reasonable to hypothesize that activation of nNOS and iNOS inside the brain is not important for the development of endotoxin tolerance.