The importance of myocardial nitric oxide (NO) production in beta-adrenergic and muscarinic signalling has been hotly debated. Conflicting data in eNOS-/- mice have been ascribed to inconsistent experimental conditions such as differences in (i) frequency of stimulation of left ventricular (LV) myocytes (e.g. a stimulation frequency of 1Hz has been associated with lower levels of myocardial NO production vs. 3 Hz), (ii) selection of control mice (wild type littermates vs. C57/BL6 mice, i.e., the conventional control strain for eNOS-/- mice), iii) the presence of LV hypertrophy in older eNOS-/- mice. Further, the recent discovery of “neuronal” NOS (nNOS) in LV myocytes has opened the possibility that this isoform may also be involved in the post-synaptic regulation of autonomic responses. The aim of this study was to dissect the contribution of myocardial constitutive NOS isoforms (eNOS and nNOS) in the inotropic response to isoprenaline (ISO, 100 nM) and ISO plus Carbachol (CCh, 1μM) in LV myocytes from humanely killed mice. Potential confounding factors such as selection of control animals and stimulation frequency were also investigated. Percentage sarcomere shortening (1 Hz or 3 Hz) was evaluated in 2-4 months old eNOS-/- or nNOS-/- mice, their respective wild type littermates (eNOS+/+ and nNOS+/+) and in C57BL/6 mice. All experiments were carried out at 35±1oC. Beta-adrenergic stimulation using ISO was followed by muscarinic agonist CCh in the continued presence of ISO. Comparisons were made using analysis of variance. All values are means±S.E.M. The inotropic response to ISO was greater in LV myocytes stimulated at 1 Hz vs. 3Hz (eNOS-/-: 0.21±0.02%, n=13 vs. 0.17±0.02%, n=13, p=0.02; eNOS+/+: 0.21±0.02%, n=13 vs. 0.13±0.01%, n=8; p=0.009), but the effect of CCh did not differ. The response to ISO alone and ISO+CCh were virtually identical in eNOS-/- and eNOS+/+ mice. Conversely, LV myocytes from C57/BL6 mice showed a significantly smaller response to ISO (C57/BL6 mice vs. both eNOS-/- and eNOS+/+ mice). nNOS-/- myocytes showed greater basal and beta-adrenergic contraction compared with both nNOS+/+ and C57/BL6 myocytes, but again the magnitude of the response to CCh was not different. There was no significant difference in heart weight: body weight ratio between any of the groups. In summary, these data demonstrate that (i) nNOS rather than eNOS-derived NO modulates beta-adrenergic inotropy in murine LV myocytes; (ii) the inappropriate use of control animals may have contributed to the conflicting data on the role of eNOS on beta-adrenergic signalling; (iii) neither eNOS nor nNOS is necessary for the cholinergic inhibition of ISO-stimulated contraction in LV myocytes.