Pleiotropic effects of serotonin (5-HT) in the cardiovascular system are well documented. However, it remains to be elucidated, whether 5-HT is present in adult mammalian cardiomyocytes. To address this issue, we investigated the levels of 5-HT in blood, plasma, platelets, cardiac tissue, and cardiomyocytes from adult mice and for comparison in human right atrial tissue. Immunohistochemically, 5-HT was hardly found in mouse cardiac tissue, but small amounts could be detected in renal preparations, whereas adrenal preparations revealed a strong positive immunoreaction for 5-HT. Using a sensitive HPLC detection system, 5-HT was also detectable in the mouse heart and human atrium. Furthermore, we could identify 5-HT in isolated cardiomyocytes from adult mice. These findings were supported by detection of the activity of 5-HT-forming enzymes-tryptophan hydroxylase and aromatic L-amino acid decarboxylase-in isolated cardiomyocytes from adult mice and by inhibition of these enzymes with p-chlorophenylalanine and 3-hydroxybenzyl hydrazine. Addition of the first intermediate of 5-HT generation, that is 5-hydroxytryptophan, enhanced the 5-HT level and inhibition of monoamine oxidase by tranylcypromine further increased the level of 5-HT. Our findings reveal the presence and synthesis of 5-HT in cardiomyocytes of the mammalian heart implying that 5-HT may play an autocrine and/or paracrine role in the heart.