In endothelial cells, two ways of endothelial nitric-oxide-synthase (eNOS)-activation are known: 1) a translocation and 2) an Akt-dependent phosphorylation of the enzyme at serin1177 (_ser1177eNOS). We have recently shown that agonist-induced _ser1177eNOS-phosphorylation also occurs in human myocardium (10). This study investigates the Ca²⁺-dependency of these two mechanisms in human atrium. Therefore, atrial tissue was obtained from patients undergoing coronary-artery bypass-operation. In immunohistochemical experiments, the translocated form of eNOS and the _ser1177phosphorylated eNOS were labelled using specific antibodies. eNOS-translocation was measured in the absence and presence of the Ca²⁺-chelator BAPTA before and after application of BRL37344 (BRL), a ₃-adrenoceptor-agonist, which increases eNOS-activity (34). In the absence of BAPTA, BRL time-dependently increased staining-intensity of translocated eNOS, whereas in the presence of BAPTA, this effect was blunted. In contrast, BRL clearly increased staining of _ser1177phosphorylated eNOS even in presence of BAPTA. This observation was confirmed by Western blot analysis. Using the NO-sensitive dye diaminofluorescein (DAF), we demonstrated that BRL induced a strong NO-release. This effect was completely abolished in the presence of BAPTA but was unaffected by LY-292004, an inhibitor of PI₃-kinase activity and eNOS-phosphorylation. Though Ca²⁺-dependent, neither the translocation of eNOS nor NO-release were changed by the adenylate-cyclase activator forskolin. Conclusions: 1) In human atrial myocardium, BRL-induced eNOS-translocation but not _ser1177eNOS-phosphorylation is dependent on intracellular Ca²⁺. 2) In atrial myocardium, eNOS-translocation and not _ser1177eNOS-phosphorylation is responsible for generating the main amount of NO. 3) Though Ca²⁺-dependent, eNOS-translocation and NO-release could not be mimicked by adenylate-cyclase activation as a mediator of -adrenergic stimulation.