SCN5A encodes the alpha-subunit of the ion channel that carries Na current in human heart. From a human cardiac cDNA library we recloned SCN5A. The new clone hH1b differed from existing clones hH1 in four and from hH1a in three positions. The common polymorphism H558R was uniquely present in hH1b. Voltage clamp study showed minor but potentially important kinetic differences between hH1b and the other clones. More dramatically, when the LQT3 mutation M1766L was introduced into the different clones, Na current was markedly reduced in the hH1 and hH1a backgrounds, whereas in hH1b the Na current was not reduced. Immunocytochemistry experiments showed a trafficking defect for M1766L Na channels in hH1 and hH1a but not in hH1b. The double-mutation M1766L/H558R in the hH1a background restored normal trafficking and current including persistent late current, suggesting the disease phenotype was the result of a "double hit" that included the common polymorphism, H558R. These results show that the choice of background clone must be carefully considered in mutagenesis studies. This also represents an example of intragenic complementation, the first for such a large protein.