We investigated the mechanism of positive inotropism of electric currents applied during the absolute refractory period. Ten Langendorff-perfused ferret hearts were instrumented to measure isovolumic left ventricular pressure (LVP) and the aequorin luminescence. Biphasic square-wave electric currents (±20 mA, total duration 30 ms) were delivered between pairs of electrodes. Six hearts were perfused at different extracellular Ca²⁺ concentrations ([Ca²⁺]_o; 1, 2, 4, and 8 mM). These signals increased LVP from 50.0 ± 9.4 to 70.1 ± 14.7, from 67.5 ± 11.0 to 79.0 ± 15.6, from 79.3 ± 21.0 to 87.1 ± 22.8, and from 84.6 ± 24.0 to 91.8 ± 28.5 mmHg at the respective [Ca²⁺]_o (P < 0.05). Peak free intracellular [Ca²⁺] ([Ca²⁺]_i) increased from 0.52 ± 0.13 to 1.37 ± 0.23, from 0.76 ± 0.23 to 1.73 ± 0.14, from 1.10 ± 0.24 to 2.05 ± 0.33, and from 1.41 ± 0.36 to 2.24 ± 0.36 µM/ml, respectively (P < 0.001). With the use of 1 mg/l propranolol with 1 mM [Ca²⁺]_o, LVP and [Ca²⁺]_i were increased significantly from 48.7 ± 8.18 to 56.3 ± 6.11 mmHg and from 0.61 ± 0.11 to 1.17 ± 0.20 µM, respectively (P < 0.05). In conclusion, positive inotropism of such electrical currents was due to increased peak [Ca²⁺]_i and Ca²⁺ responsiveness of the myofilaments did not change significantly.