We have previously shown a time-of-day variation in the response of systolic [Ca2+]i to the non-specific β-adrenergic (β-ADR) agonist isoproterenol (ISO), linked to a variation in Nitric Oxide (NO) signalling [1]. This may reflect stimulation of β3-ADRs, which induces a NO-dependent negative inotropic response [2]. As the action potential duration (APD) regulates systolic [Ca2+]i, and a time-of-day variation exists in the cardiac action potential [3], we set out to investigate the effect of β3-ADR stimulation and NO-signalling on the APD and systolic [Ca2+]i. Ventricular myocytes were isolated by enzymatic digestion at time points corresponding to 3 hours into the male Wistar rats rest (ZT3) and active-period (ZT15). Measurement of systolic [Ca2+]i was made in myocytes loaded with Fura-2 and APD using the whole-cell patch clamp technique. A significant time-of-day variation was found in systolic [Ca2+]i following stimulation with ISO (10nM), a non-specific β-ADR agonist, which was higher in ZT3 (1040.0 + 116.9nM) compared to ZT15 myocytes (428.0 + 63.1nM) (n=3, S.E.M., 2-way ANOVA, P<0.001). The difference in systolic [Ca2+]i during ISO stimulation was abolished following inhibition of NOS with L-NNA (500µM) (2-way ANOVA, ns). To determine whether this time-of-day variation in response to ISO can be explained by a variation in AP configuration in response to β-ADR stimulation, APD at 30% (APD30) and 50% (APD50) were recorded. ISO stimulation increased APD30 and APD50 significantly more in ZT15 than ZT3 myocytes, with % increase in APD30 of 120.3 + 14.9% in ZT15 compared to 10.6 + 8.2% in ZT3 myocytes (n=3, S.E.M., students t-test, P<0.001), and APD50 of 95.9 + 13.2% in ZT15 compared to 11.6 + 7.4% in ZT3 myocytes (n=3, S.EM., students t-test, P<0.001). We also investigated systolic [Ca2+]i and APD in ZT3 and ZT15 myocytes in response to the specific β3-ADR agonist BRL37344 (200nM), to determine if time-of-day variation in systolic [Ca2+]i following ISO-stimulation could be explained by variation in β3-ADR signalling. A significant reduction in systolic [Ca2+]i in ZT3 myocytes was found following BRL37344 stimulation, from 458.5 + 41.2nM to 361.2 + 18.0nM (n=4, 2-way ANOVA, P<0.001) but no effect on ZT15 myocytes. BRL37344 also significantly reduced APD30, (18.3 + 2.2ms to 14.4 + 1.6ms) (n=5, 2-way ANOVA, P<0.001), and APD50 (32.9 + 4.3ms to 26.5 + 3.1ms) (n=5, 2-way ANOVA, P<0.001) in ZT3 myocytes with no significant change in ZT15 myocytes. Our data shows a reduction in systolic [Ca2+]i in rest-period myocytes (ZT3) in response to β3-ADR stimulation, which may reflect the reduction in APD. This suggests that the reduced response of systolic [Ca2+]i to ISO-stimulation in active-period myocytes (ZT15) is not due to a strong negative inotropic action of β3-ADR activation during the active period.