β3-adrenergic receptor (AR) stimulation elicits a NO-dependent negative inotropic effect in murine and human LV myocardium, which has been attributed to coupling of β3-AR with endothelial nitric oxide synthase (eNOS). It is now well established that a “neuronal” NOS (nNOS) is also constitutively expressed in mammalian LV myocytes where it contributes to the regulation of basal and beta-adrenergic contractility. Here we tested whether nNOS contributes to the β3-AR mediated decrease in contraction and peak intracellular Ca transient in murine LV myocytes from humanely killed mice. Cell shortening (1Hz) was evaluated in myocytes isolated from eNOS-/- and nNOS-/- mice. Intracellular Ca transient (F365/F380 of Fura-2) was investigated in nNOS-/- and nNOS+/+ myocytes. The partially selective β3-adrenoceptor agonist, BRL-37344 (BRL, 10 μM) was used in conjunction with the β1 and β2-AR blocker, nadolol (10 μM). All experiments were carried out at 35±1oC. Immunoblots for eNOS and nNOS were obtained from LV homogenates from nNOS-/- and eNOS-/- mice, respectively. Comparisons were made by using the unpaired t test. All values are means±S.E.M. Application of BRL + nadolol for 3-5 minutes decreased cell shortening in LV myocytes isolated from control mice (from 6.17 ±0.7% to 5.18 ± 0.7%, n=12, P<0.01) whereas nadolol alone had not effect on cell shortening (n=7, P=NS). eNOS gene deletion abolished the negative inotropic effect of BRL+nadolol (from 5.99±0.45 % to 6.24±0.5%, n=19, P=NS). Interestingly, the decrease in cell shortening in response to BRL+nadolol was also abolished in nNOS-/- myocytes (from 5.92±0.28% to 5.69±0.26%, n=45; P=NS). Consistent to this result, BRL+nadolol significantly decreased the amplitude of Ca2+ transient in control LV myocytes but not in nNOS-/- mice (F365/F380: from 0.58±0.04 to 0.56±0.03, n=27, P=NS). The myocardial nNOS protein level was not altered in eNOS-/- mice and vice versa (n=4 in each group). In summary, our data indicate that both myocardial constitutive NOS isoform are required for the negative inotropic effect of β3-AR stimulation to occur.