Several studies have demonstrated an association between Brugada syndrome (BrS) and mutations in genes encoding ion channel subunits including SCN5A, CACNA1C, CACNB2b, SCN1B, and KCNE3. Mutations in SCN5A, encoding the voltage-gated sodium channel Nav1.5, represent the majority with greater than 293 mutations in SCN5A linked to the syndrome (Kapplinger et al, Heart Rhythm, In press 2009). We identified a missense mutation (G1408R) in SCN5A in a large BrS family. Intriguingly, this mutation had been reported earlier in two independent studies and has also shown to be associated with Sick Sinus Syndrome (SSS) and Cardiac Conduction Defect (CCD) (Kyndt et al, 2001; Benson et al, 2003). Nav1.5-G1408R channels heterologously expressed in CHO cells and studied using patch-clamp techniques failed to generate any sodium channel current (INa). Co-expression of the mutated channels with wild-type (WT) channels resulted in a 50% reduction of current amplitudes with no changes in kinetic properties when compared with WT channels. The residue resides in the DIII pore region and is conserved among species. We addressed the importance of this amino acid at position 1408 by replacing it with another small neutral amino acid (G1408A) and by substituting a negatively charged aspartic acid (G1408D). Our results show that substituting glycine with alanine retains WT behavior while exchange to the positively charged arginine (G1408R) or negatively charged aspartic acid leads to a complete loss-of-function. In conclusion, we describe a SCN5A mutation associated with BrS which results in a loss of function of INa important for action potential generation. Further, we show that the presence of a neutral hydrophobic amino acid at this position is crucial for normal channel function.