We have previously shown that the transmembrane segment plus either the extracellular or intracellular domain of the β1 subunit are required to modify cardiac Nav1.5 channels. In this study, we coexpressed the intracellular domain of the β2 subunit in a β1/β2 chimera with Nav1.5 channels in Xenopus oocytes and obtained an atypical recovery behavior of Nav1.5 channels not reported before for other Na+ channels: Recovery times of up to 20ms at −120mV produced a similar fast recovery as observed for Nav1.5/β1 channels, but the current amplitude decreased again at longer recovery times and reached a steady-state level after 1–2s with current amplitudes of only 43±2% of the value at 20ms. Current reduction was accompanied by slowed inactivation and by a shift of steady-state activation toward depolarized potentials by 9mV. All effects were reversible and they were not seen when deleting the β2 intracellular domain. These results describe the first functional effects of a β2 subunit region on Nav1.5 channels and suggest a novel closed state in cardiac Na+ channels accessible at hyperpolarized potentials.