A third cardiac β-adrenoceptor (AR) has been reported in ventricular cells from a number of species including humans (Gauthier et al., 1998). This receptor has been shown to mediate negative inotropic effects unlike the other β-ARs of the heart. BRL37344 is a β3 agonist and upon its stimulation of the β3-AR, the Gi sub-unit is activated which leads to production of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS). The production of NO activates guanylate cyclase (GC) which catalyses the production of cGMP. An increase in cGMP levels activates protein kinase G (PKG) and calcium (Ca2+) entry via the L-type Ca2+ channel is inhibited hence causing the cardioinhibitory effects. Due to the negative inotropic effect of BRL37344, investigations of its expression during heart failure have been carried out. It has been shown that these receptors are up-regulated in such circumstances to protect the heart from excess catecholamines (Moniotte et al., 2001). In this study we have used BRL37344 in quiescent WKY rat atrial cells to investigate its effect on Ca2+ spark frequency. Atrial cells from WKY rats were loaded with fluo-4AM (5µM) and whole cell and line scan mode images of Ca2+ events within these cells were obtained every 3ms using an LSM 510M confocal microscope. Cardiomyocytes were perfused with BRL37344 (300nM) in the presence and absence of a β-blocker, propranolol (200nM). It was discovered that the frequency of Ca2+sparks decreased significantly in the presence of BRL37344 (and propranolol) compared to propranolol on its own (15.4 ± 3.3 sparks s-1 compared to 29.3 ± 6.4 sparks s-1 respectively (n = 6, p = 0.05). No larger Ca2+ events such as non-propagating wavelets or whole cell waves were observed. Furthermore, when the quiescent atrial cells were perfused with only BRL37344 the mean frequency of Ca2+ sparks tended to decrease compared to basal conditions (14.5 ± 2.4 spark s-1 to 22.0 ± 3.0 sparks s-1 respectively (n = 5, p = ns). Furthermore, atrial cells under basal conditions expressed on average 1.07 ± 0.24 wavelets s-1. The reduction observed in intracellular Ca2+ release events with BRL37344 was due to β3-ARs stimulation, mediated by a PKG-dependent pathway. The negative inotropic effect of BRL37344 observed in rat atrial cells is associated with fewer spontaneous Ca2+ release events.