Over 10% of the rat cardiac genes exhibit diurnal variations, and cycling of metabolic genes link metabolic activity and oxygen consumption with peak power. We have demonstrated that key elements of E-C coupling in rat ventricular myocytes show strong diurnal rhythms, including their response to the non-specific β-adrenoreceptor agonist isoproterenol, which stimulates β1, 2 and 3-receptors. As β3-adrenoreceptor stimulation involves activation of nitric oxide (NO)-dependent pathways through coupling to nitric oxide synthase (NOS), we looked at the involvement of NOS in the diurnal variation of E-C coupling (Massion & Balligand, 2003; Seddon et al., 2007). Adult male Wistar rats were housed with a 12 hour light/dark cycle. Hearts were excised from animals at two opposing timepoints of ZT3 and ZT15, where ZT0 refers to “lights on”, and single left ventricular myocytes were isolated by enzymatic digestion. Measurements of [Ca²⁺]_i were made using Fura-2 and L-type Ca²⁺ channel density was determined electrophysiologically using the whole cell, patch clamp technique. We looked at the modulating effect of the non-specific NOS inhibitor Nω-Nitro-L-arginine (L-NNA) on systolic [Ca²⁺]_i and L-type Ca²⁺-current density at rest and during β-adrenergic stimulation with isoproterenol. L-NNA (500μM) had no significant effect on the basal systolic [Ca²⁺]_i and L-type Ca²⁺-current recorded from ZT3 or ZT15 myocytes in normal Tyrode. However, following stimulation with 5nM isoproterenol, L-NNA had a stimulatory effect on both systolic [Ca²⁺]_i and L-type Ca²⁺-current density. This stimulation was greater in ZT15 compared to ZT3 myocytes, with an increase in systolic [Ca²⁺]_i of 32.6 ± 7.5% (n=29) in ZT3 myocytes vs. 68.8 ± 9.9% (n=46) in ZT15 myocytes (S.E.M; students t-test, P<0.05) and an increase in current density of 2.7 ± 2.4% (n=12) in ZT3 myocytes vs. 15.4 ± 2.6% (n=13) in ZT15 myocytes (S.E.M; students t-test, P<0.01). Quantitative RT-PCR analysis of snap frozen left ventricular tissue revealed no significant difference in expression levels of the L-Type Ca²⁺-channel (cacna1c) or eNOS between time points. However, we detected a significant 5-fold increase in nNOS mRNA levels in ZT15 over ZT3 myocytes (S.E.M; n=4, students t-test on ΔCt values, P<0.05). Our data shows that the diurnal variation in the sensitivity of E-C coupling of rat ventricular myocytes to stimulation with isoproterenol, and this appears to involve NOS.