—The congenital long-QT syndrome (LQT), an inherited cardiac arrhythmia characterized in part by prolonged ventricular repolarization, has been linked to 5 loci, 4 of which have been shown to harbor genes that encode ion channels. Previously studied LQT-3 mutations of SCN5A (or hH1), the gene that encodes the human Na⁺ channel α-subunit, have been shown to encode voltage-gated Na⁺ channels that reopen during prolonged depolarization and hence directly contribute to the disease phenotype: delayed repolarization. Here, we report the functional consequences of a novel SCN5A mutation discovered in an extended LQT family. The mutation, a single A→G base substitution at nucleotide 5519 of the SCN5A cDNA, is expected to cause a nonconservative change from an aspartate to a glycine at position 1790 (D1790G) of the SCN5A gene product. We investigated ion channel activity in human embryonic kidney (HEK 293) cells transiently transfected with wild-type (hH1) or mutant (D1790G) cDNA alone or in combination with cDNA encoding the human Na⁺ channel β₁-subunit (hβ₁) using whole-cell patch-clamp procedures. Heteromeric channels formed by coexpression of α- and β₁-subunits are affected: steady-state inactivation is shifted by –16 mV, but there is no D1790G-induced sustained inward current. This effect is independent of the β₁-subunit isoform. We find no significant effect of D1790G on the biophysical properties of monomeric α- (hH1) channels. We conclude that the effects of the novel LQT-3 mutation on inactivation of heteromeric channels are due to D1790G-induced changes in α- and β₁-interactions.