Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia associated with substantially increased morbidity and mortality. Growing evidence strongly implicates hereditary determinants in the pathogenesis of AF. However, AF is genetically heterogeneous and the genetic defects responsible for AF in the majority of cases remain to be identified. In this study, all the coding exons and splice junctions of GATA4, a gene encoding a zinc-finger transcription factor critical for normal cardiac morphogenesis, were sequenced in a cohort of 150 unrelated patients with idiopathic AF. The available relatives of the mutation carriers and a total of 200 unrelated ethnically matched healthy individuals used as controls were genotyped for the presence of mutations identified in index patients. The functional effect of the mutant GATA4 was characterized using a luciferase reporter assay system. As a result, two novel heterozygous GATA4 mutations (p.Y38D and p.P103A) were identified in 2 unrelated families with AF, respectively. In each family the mutation co-segregated with AF and was absent in the 400 control chromosomes. Functional analysis showed that the mutations of GATA4 were associated with a significantly decreased transcriptional activity. The findings expand the mutation spectrum of GATA4 linked to AF, and further support the notion that compromised GATA4 confers genetic susceptibility to AF.