The endothelial type nitric oxide synthase (NOS) termed as NOS-III has been demonstrated in various nonendothelial cells. Although NOS-III was recently found in the cardiac tissues, identification of the cells expressing NOS-III gene in the heart remains unknown. In this study, we compared the expression of NOS-III mRNA in various tissues of rats by Northern blot analysis using a homologous cDNA probe and identified the cell type by in situ hybridization histochemistry with a cRNA probe. NOS-III mRNA was found more abundantly in atria and ventricle than in other various tissues examined. NOS-III mRNA was intensely and specifically visualized in the cardiac myocytes as well as in the endothelial cells of the heart. No staining was seen with a sense cRNA probe. These results provide the first in vivo evidence for NOS-III mRNA in the cardiac myocytes and suggest its role in the regulation of cardiac functions.