We have found a novel nonsense mutation in the C-terminus of HERG in a four-generation Chinese family with long QT syndrome and investigated the molecular mechanism of this mutation in vitro. Six family members, including the proband, were clinically affected. Syncope and ventricular tachycardia of torsades de pointes were triggered by startling or emotional stress, and beta-adrenergic blockade treatment was ineffective. Haplotype analysis showed that only LQT2 markers cosegregated with the disease, and sequence analysis revealed a substitution of T with C at nucleotide position 2770 of the HERG gene (U04270), which creates a stop codon at amino acid position 863 (R863X) of the HERG protein, leading to a deletion of 296 amino acids. Whole cell patch clamp studies showed that the R863X HERG could not induce time-dependent current. Coexpression of R863X with wild-type HERG showed reduced current densities and accelerated voltage-dependent inactivation of HERG channels. Subcellular localization of R863X-EGFP revealed that the mutant did not traffic to the cell surface. These data suggest that R863X failed to form functional HERG channels, contributing to a prolongation of the QT interval and long QT syndrome with a dominant phenotype. These findings provide new insights into the structure-function relationships of the HERG C-terminus.