We have previously shown that the negative inotropic response of the β3-adrenergic receptor (β3-ADR) specific agonist BRL37344 was associated with a reduced systolic [Ca2+]i and SR Ca2+-content ([Ca2+]SR) which may have resulted from the shortening of action potential duration (APD). This negative inotropic action of β3-ADR has been linked by other groups to Nitric Oxide (NO) signalling [1]. As β3-ADR signalling is considered to be cardio-protective against Ca2+-overload injury [2] we set out to investigate a possible anti-arrhythmic effect of β3-ADR stimulation. Ventricular myocytes were isolated by enzymatic digestion of male Wistar rat hearts. Arrhythmic activity was determined from video images of 20-30 myocytes electrically stimulated at 1Hz with high concentrations of β1-ADR agonist. [Ca2+]i was measured in myocytes loaded using Fura-2 and APD using whole-cell patch-clamp recording. Data shown as number of hearts (number of experiments), mean ± S.E.M., analysed via one-way ANOVA, with a Tukey’s post hoc test. To determine the anti-arrhythmic effect of β3-ADR stimulation, arrhythmic activity was induced by the β1-ADR specific agonist dobutamine (1µM for 5 minutes). Pre-treatment of cells with BRL37344 (200nM for 5 minutes) resulted in a significant reduction in the percentage of cells displaying arrhythmic activity in dobutamine from 34.5 ± 2.7% vs 5.5 ± 2.6% (n=4 (8), p<0.0001). To determine whether the reduction in arrhythmic activity by BRL37344 was due to a modulation of the increase in APD by dobutamine, APD at 30% (APD30), 50% (APD50) and 90% (APD90) were recorded. Dobutamine resulted in a significant lengthening of the APD30 from 13.97 ± 1.53ms to 23.99 ± 1.94ms (n=4-6 (15-16), p<0.01) and APD50 from 22.83 ± 2.28ms to 41.15 ± 2.48ms (n=4-6 (15-16, p<0.001). This lengthening of ADP30/50 was not significantly altered by cells pre-treated with BRL37344. As BRL37344 reduced systolic [Ca2+]i and SR Ca2+-loading, we looked at the effects of BRL37344 pre-treatment on calcium regulation in response to dobutamine. Pre-treatment of cells with BRL37344 did not cause any significant change in the dobutamine-induced increase in systolic [Ca2+]i or [Ca2+]SR. However, BRL37344 pre-treatment resulted in a significant change in the dobutamine-induced reduction in the exponential time constant of the Ca transient, from 289.40 ± 20.71ms to 121.40 ± 3.59ms vs 102.50 ± 6.29ms in pre-treated cells (n=3-4 (14-22),p<0.05), indicative of an additional increase in SERCA-2A activity beyond the dobutamine-induced increase in activity. Our data suggests that β3-ADR pre-treatment causes an additional increase in the activity of SERCA-2A beyond the level of β1-ADR stimulation alone. As a result calcium is more rapidly sequestered to the SR during relaxation, removing the burden of Ca extraction from the Sodium-Calcium exchanger (NCX), potentially reducing the inward sodium current during cardiomyocyte relaxation.