Objective: Ventricular repolarization is promoted by the delayed rectifier potassium currents. Mutations in the genes underlying these currents, primarily being I _Kr, I _Ks, and I _K1, have previously been found to cause congenital long-QT syndrome (LQTS). Kir3.1/3.4 channels previously been ascribed a prominent role in atrial and nodal parasympathetic regulation, but a functional role in ventricle has not previously been demonstrated in humans. The purpose of this study was a functional assessment of a mutation in Kir3.4 identified in a large LQTS family. Methods: A large Chinese family (4 generations, 49 individuals) with an autosomal dominant trait of LQTS was clinically evaluated. Genome-wide linkage analysis was performed using polymorphic microsatellite markers to map the locus, and the positional candidate genes were screened by sequencing for mutations. The expression pattern and functional characteristics of the mutated protein was investigated by Western blotting and patch-clamp electrophysiology. Results: The genetic locus of the LQTS-associated gene was mapped to chromosome 11q23.3-24.3, where a heterozygous mutation in the gene KCNJ5, coding for the G protein-coupled inward rectifier potassium channel subunit Kir3.4, was identified (Kir3.4-G387R). This specific mutation was present in all 10 affected family members, but absent in 528 ethnically matched controls and in the healthy family members. A cross-species alignment of Kir3.4 protein sequences displayed that the altered amino acid is highly conserved evolutionarily. Heterologous expression studies revealed a loss-of-function phenotype of Kir3.4-G387R caused by reduced surface membrane expression. Western blotting of human cardiac tissue demonstrated significant expression of Kir3.4 in ventricle. Conclusion: We provide evidence for Kir3.4 being associated with LQTS, and thereby reveal a physiological function of Kir3 channels in human ventricles.