Atrial natriuretic peptide (ANP) which is produced by mammalian cardiomyocytes is involved in the regulation of blood pressure and fluid homeostasis. The peptide induces suppression of the renin-angiotensin as well as the sympathetic nervous systems to protect cardiovascular homeostasis, which is also deteriorated by the stress. The present study was designed to detect ANP and to examine expression of ANP mRNA in all heart compartments. Additionally, we investigated whether ANP distribution and ANP mRNA expression in the heart could be affected by the stress. We used male Sprague-Dawley (SD) rats (200g). Treatment of animals was in accordance with the Declaration of Helsinki Guiding Principles on Care and Use of Animals [DHEW Publication, NHI 80-23]. The study was approved by the Ethical Review Committee, First Faculty of Medicine, Charles University in Prague. A restraint stressor (immobilization, IS) and IS combined with partial immersion of rats into water (ICS) were applied to animals for one hour. One or three hours after the stress termination, the rats were decapitated, hearts were removed, and each atrium and ventricle were examined separately. Thus, all studies were performed in five groups of animals: in controls, IS1, IS3, ICS1, and ICS3 (n=4-6 per group). ANP localization in sections of preparations was identified by indirect immunohistochemistry with the use of specific commercial antibody. Expression of ANP mRNA was detected by real-time qRT PCR in all heart chambers by comparing their threshold cycle values (CT) to CT of reference gene β-actin. The relative expression ratios were calculated using Mann-Whitney test. Values are means ± S.E.M. In control rats, ANP-immunopositivity was strongly expressed in the left (LA) and right (RA) atrium in coarse granules within the cytoplasm (n=3 per group). No immunofluorescence was observed in the left (LV) and right (RV) ventricle in both control and stressed animals. ANP mRNA expressions were much higher in atria than in ventricles. In control rats, rank order of the gene expression was: LA = 138.8 (p<0,005) > RA = 39.2 (p<0,005) > RV = 10.1 (p<0,005) > LV = 1. The stress exposition led to increased expression of ANP mRNA by 56% (1.56±0.18, p<0.05) in RA, 22% (1.22±0.09, p<0.05) in LA, and 90% (1.90±0.37, p<0.05) in LV after IS1. The increase was also observed after IS3 in the same compartments, by 53% (1.53±0.14, p<0.05) in RA, 49% (1.49±0.21, p<0.05) in LA, and 134% (2.34±0.20, p<0.05) in LV. Combined stress (ICS3) caused the increase of ANP mRNA expression by 67% (1.67±0.22, p<0.05) in LA and by 187% (2.87±1.0, p<0.05) in LV. In conclusion, restraint stressors induced changes in expression of ANP mRNA in cardiomycytes of atria and left ventricles; these effects may have relevance to ANP induced cardioprotection.