Our laboratory has recently detected mRNA of thyrotropin-releasing hormone (TRH) in the rat heart. The density of mRNA for TRH is five-fold higher in the atria than in the left and right ventricle. We also found TRH receptor mRNA and 3H-TRH-binding sites in both ventricles. Cardiac contractility was stimulated after intracoronary administration of TRH. This study was performed to investigate the localization of TRH in the heart. We utilized in situ hybridization histochemistry (ISHH) to localize TRH mRNA expression in the rat heart. ISHH was performed on fresh frozen heart tissue sections which were hybridized with a specific 35S-TRH oligo probe and subsequently processed by autoradiography. The autoradiographic signals corresponding to TRH mRNA were analyzed with an image program. For positive controls TRH mRNA was identified in the hypothalamic paraventricular nucleus. This test confirms the specificity of the TRH oligo probe. Cardiac hybridization signals were observed predominantly in the atria and localized preferentially in atrial connective tissues, vascular adventitia and atrial cardiomyocytes. No hybridization signals were found in ventricular cardiomyocytes. These observations suggest that TRH is synthesized in atrial myocytes and atrial vascular structures. Based on studies which show synthesis of the TRH receptors in ventricular cardiomyocytes, we hypothesize that atrial TRH is an endocrine source for the stimulation of ventricular contractility and that endothelial and adventitial TRH may play a role(s) in the regulation of the growth and/or vasomotor tome of the cardiac vascular system.