The microinjection of carbachol, a cholinergic agonist, into the medial septal area (MSA) induces anti-diuresis and increase in arterial pressure which is respectively due to vasopressin release and/or increase in sympathetic nerve activity (SNA). MSA projects directly to the paraventricular nucleus of hypothalamus (PVN), an important forebrain area involved in vasopressin release and SNA regulation. In the brain, reactive oxygen species (ROS) may modulate autonomic and behavioral responses. Previously, we have demonstrated that hydrogen peroxide (H2O2) microinjected into the MSA reduced the antidiuresis and the pressor response induced by carbachol into the same area. In the present study we investigate the effects of carbachol combined or not with H2O2 into the MSA on c-FOS expression in vasopressinergic cells of the PVN. Male Holtzman rats (280-320 g) were anesthetized with ketamine (80 mg kg-1) combined with xylazine (7 mg kg-1, both i.p.) and implanted with stainless steel guide-cannulas into the MSA.The Ethics Committee for Animal Care and Use of the Dental School of Araraquara, UNESP, approved the experimental protocols used in the present study (Proc. CEUA 02/2012). Seven days later, rats were randomly assigned for 3 groups (n = 3/group): PBS into ASM + saline into ASM; PBS into ASM + carbachol into MSA and H2O2 into MSA + carbachol into MSA. H2O2 (2.5 µmol/ 0.5 µl) or PBS (0.5 µl) was injected into MSA 1 minute before of the injection of carbachol (4 nmol/0.5 µl) in the MSA. After 90 min, rats were deeply anesthetized with sodium thiopental (50 mg/kg, ip), perfused with 4% paraformaldehyde and sections of PVN underwent double-staining immunohistochemistry for c-FOS and AVP using DAB staining with 1% cobalt chloride and 1% nickel ammonium sulfate to intensify the cell nucleus. Values are means ± S.E.M., compared by ANOVA. Carbachol into the MSA increased c-FOS expression on parvocellular (pPVN) (24.2 ± 4.2, vs. PBS + saline: 0.2 ± 0.1 cells/section, p < 0.05) and on magnocellular (mPVN) vasopressinergic cells (98.5 ± 5.9, vs. PBS + saline: 1.1 ± 0.4 cells/section, p<0.05). Previous injection of H2O2 reduced the double- labeling on both pPVN (12.7 ± 4.6 cells/section, p<0.05 compared to PBS + carbachol) and mPVN (46.4 ± 11.2 cells/section, p<0.05 compared to PBS + carbachol). These data show that the pre-treatment with H2O2 into the MSA decreases the activation of PVN vasopressinergic cells induced by carbachol into the same area, which could explain the reduction of the anti-diuresis and in the increase of arterial pressure induced by carbachol into the MSA in H2O2 pre-treated rats.