Emerging evidence suggests that a neuronal nitric oxide synthase (nNOS), which has been located to the cardiac sarcoplasmic reticulum, regulates Ca2+-handling and contraction in normal and failing hearts. Recently, data from our group showed that both basal relaxation and the lusitropic effect of β-adrenergic stimulation were impaired in LV myocytes of nNOS knock out mice (nNOS-/-). The decay of the [Ca2+]i transient was also slower in nNOS-/- LV myocytes suggesting that sarcoplasmic Ca2+-ATPase (SERCA) activity may be depressed in this strain. Phospholamban (PLB) phosphorylation at Ser16 and Thr17 is known to be responsible for increasing SERCA activity both in basal conditions and in response to beta-adrenergic stimulation. The aim of present study was to investigate whether a reduction in Ser16- and Thr17- phosphorylation of PLB in the nNOS-/- LV myocardium may account for the impaired basal and beta-adrenergic relaxation in these mice. Mice were humanely killed and western blots performed on LV homogenates or LV myocyte membrane subfractions using specific antibodies to the following: Phospholamban (Affinity Bioreagents), SERCA2a (Affinity Bioreagents), Ser16 and Thr17 phosphorylation of Phospholamban (Cyclacel). The level of expression was normalized to GAPDH and comparisons were made by using the unpaired t test. All values are means±S.E.M. SERCA2a protein level was unchanged in the LV myocardium of nNOS-/- mice (SERCA2a/GAPDH ratio: 2.4 ± 0.2 vs. 1.9 ± 0.3 in nNOS+/+, n=5, P=NS). Total PLB, however, was modestly but significantly decreased in nNOS-/- (PLB/GAPDH ratio: 0.7 ± 0.005 vs. 0.5 ± 0.02 in nNOS+/+, n=3, p<0.05). These changes resulted in a modest increase in SERCA2A/PLB ratio, which should facilitate rather than depress relaxation. However, further experiments showed that the Ser16- and Thr17-phosphorylated fractions of PLB were significantly lower in the nNOS-/- LV myocardium (PLB Ser16/GAPDH ratio: 0.8 ± 0.04 vs. 2.8 ± 0.03 in nNOS+/+, n=5, p<0.001; PLB Thr17/GAPDH ratio: 0.4 ± 0.03 vs. 0.6 ± 0.01 in nNOS+/+, p<0.05). Pre-incubation of LV myocytes with isoprenaline (ISO, 1 μM) resulted in a significant increase in the Ser16-phosphorylated fraction (n=3) and to a much lesser extent in Thr17-phosphorylation (n=3), however, both phosphorylated fractions remained significantly lower in nNOS-/- myocytes. Co-immunoprecipitation of nNOS with Ser16 PLB suggests that there might be a physical interaction between these proteins. In summary, these findings demonstrate a previously unrecognized role for nNOS in regulating PLB phosphorylation and hence basal and beta-adrenergic myocardial relaxation.