Variable transcription of the cardiac sodium channel gene is a candidate mechanism determining arrhythmia susceptibility. We have previously cloned and characterized the core promoter and flanking region of SCN5A, encoding the cardiac sodium channel. Loss-of-function mutations in this gene have been reported in approximately 20% of patients with Brugada syndrome, an inherited cardiac electrical disorder associated with a high incidence of life-threatening arrhythmias. In this study, we identified DNA variants in the proximal 2.8 kb promoter region of SCN5A and determined their frequency in 1,121 subjects. This population consisted of 88 Brugada syndrome patients with no SCN5A coding region mutation, and 1,033 anonymized subjects from various ethnicities. Variant promoter activity was assayed in CHO cells and neonatal cardiomyocytes by transient transfection of promoter-reporter constructs. Single-nucleotide polymorphisms (SNPs) were identified at approximately 1/200 base pairs which are: 11 in the 5'-flanking region, 1 in exon 1, and 5 in intron 1. In addition, a haplotype consisting of two SNPs in complete linkage disequilibrium was identified. Minor allele frequencies were >5% in at least one ethnic panel at 5/19 polymorphic sites. In vitro functional analysis in cardiomyocytes identified four variants with significantly (P<0.05) reduced reporter activity (up to 63% reduction). The largest changes were seen with c.-225-1790 G>A, which reduced reporter activity by 62.8% in CHO cells and 55% in cardiomyocytes. From these results, we can conclude that the SCN5A core promoter includes multiple DNA polymorphisms with altered in vitro activity, further supporting the concept of interindividual variability in transcription of this cardiac ion channel gene.