To evaluate the effects of liothyronine (3,5,3'-triiodo-L-thyronine, T₃) on Na⁺ channel current (I_Na) properties, I_Na was recorded in adult guinea pig ventricular myocytes. T₃ (1 nM) acutely increased whole cell I_Na and shifted the steady-state I_Na inactivation curve dose dependently. When the pipette solution contained 100 µM GTP or GTPS, the effect of T₃ on the whole cell I_Na was increased two- to threefold. This effect was almost completely abolished by pertussis toxin preincubation. In the cell-attached patch, T₃ increased the open probability of single I_Na by reducing the null probability. In the inside-out patch, T₃ effect was 10 times faster than that in whole cell and cell-attached patches while GTPS was present and could be completely washed out. T₃ alone slightly increased the channel open probability by increasing the closed state to open state rate constant (k_CO) and reducing the null probability. GTPS exposure only increased the number of functional channels. T₃ and GTPS synergistically enhanced the channel open probability 5.8 ± 0.5-fold by increasing k_CO, decreasing the open state to absorbing inactivated state rate constant, and greatly reducing the null probability. These results demonstrate that T₃ acts on the cytosolic side of the membrane and acutely activates I_Na. Pertussis toxin-sensitive G protein modulation greatly magnifies the T₃ effects on the channel kinetics and null probability, thereby increasing the channel open probability.