A negative staircase response is associated with a high intracellular Na concentration in human and animal myocardium (Shattock & Bers, 1989). The mechanisms underlying this phenomenon remain unclear but has been postulated to involve the relative phosphorylation of intracellular sites. β-agonists act via protein kinase-A (PKA), and such sites in turn can be a target for the Ca-dependent phosphatase, calcineurin. The potential role of PKA and calcineurin in modifying the staircase response was explored by examining the effects of the calcineurin inhibitor cyclosporin-A, alone or in combination with isoprenaline. Isolated mouse left ventricles strips (<1 mm diam) were superfused with Tyrode’s solution (24 mM NaHCO₃/5% CO₂, pH 7.40, 37°C), attached to an isometric force transducer and field-stimulated between 0.1-6.0 Hz (values normalised to 1 Hz values=100%). Interventions, isoprenaline (10 μM + 10 μM ascorbic acid) and the calcineurin inhibitor cyclosporin A (10 μM) were added to the superfusate. Data are expressed as mean ± S.E.; *p<0.05; **p<0.01; ***p<0.001 by paired Students’s t-tests. Multiple group comparisons used ANOVA. An increase of stimulation frequency decreased significantly isometric peak tension across the range: e.g. at 0.4, 1.2, 2.0 and 6.0 Hz (n=7) normalized tensions were 136.0±12.2%*; 93.6±1.6%**; 74.9±6.7%***, 64.4±15.6%*, respectively. Superfusion with the β-adrenergic agonist, isoprenaline (Iso) generated a positive inotropic effect at all frequencies. However, the negative staircase associated with increasing frequency was abolished: e.g. values at 0.4, 1.2, 2.0 and 6.0 Hz (n=7) were not significantly different from each other: 156.9±21.4%; 134.5±25.0%; 118.3±19.4%, 150.2±32.4%, respectively. A quantitative index of the force-frequency relationship calculated the tension ratio at 1.2 and 0.4 Hz (T_1.2/T_0.4). Isoprenaline increased T_1.2/T_0.4 from 0.64±0.04 to 0.86±0.22*, n=6. Pretreatment with cyclosporin A (CysA) alone for 15 min did not affect T_1.2/T_0.4 (0.61±0.07; 0.70±0.06; control vs CysA). Moreover, the combination of CysA and Iso (0.81±0.05; n=5) was not different from the action of Iso alone. Our data demonstrate a negative force-frequency relationship in mouse myocardium over a wide range of frequencies, which was flattened with isoprenaline. This action of isoprenaline was unaffected by CysA. We hypothesise that isoprenaline and calcineurin act at different intracellular phosphorylation sites.