Physostigmine (eserine) is the methyl carbamic ester of phenolic trimethyl ammonium compound. It is a powerful inhibitor of acetylcholinesterase enzyme activity and used in some therapeutic preparation. In this work, experiments were carried out in-vivo to study the intraperitoneal (i.p.) infusion of eserine on the activity of nitric oxide synthase (NOS) enzyme, obtained from whole and five different parts of rat brain, namely: basal ganglia, frontal cortex, medulla oblongata, pons, and cerebellum. In this work ,two experiments were carried out. Experiment A: to study the dose dependence of i.p. infusion of eserine on NOS activity. Experiment B: to study the time dependence post-infusion of constant dose of eserine (the dose which caused 50% inhibition of the enzyme activity: I₅₀). The results showed that the inhibition of the enzyme occurred in each part studied, and the inhibition increased with increasing the infused dose of eserine, and the time post i.p. infusion, i.e., the inhibition is dose and time dependent. The highest inhibition occurred in the pons and medulla oblongata  extracts; these parts are responsible for the reflex centers of cough and vital centers.