Persistent Na+ currents are thought to be important for integration of neuronal responses. Here, we show that betagamma subunits of G proteins can induce persistent Na+ currents. Coexpression of G beta2gamma3, G beta1gamma3, or G beta5gamma3, but not G beta1gamma1 subunits with rat brain type IIA Na+ channel alpha subunits in tsA-201 cells greatly enhances a component of Na+ current with a normal voltage dependence of activation but with dramatically slowed and incomplete inactivation and with steady-state inactivation shifted +37 mV. Synthetic peptides containing the proposed G betagamma-binding motif, Gln-X-X-Glu-Arg, from either adenylyl cyclase 2 or the Na+ channel alpha subunit C-terminal domain reversed the effect of G beta2gamma3 subunits. These results are consistent with direct binding of G betagamma subunits to the C-terminal domain of the Na+ channel, stabilizing a gating mode responsible for slowed and persistent Na+ current. Modulation of Na+ channel gating by G betagamma subunits is expected to have profound effects on neuronal excitability.