Previous studies suggest native cardiac IKr channels are composed of alpha subunits encoded solely by the 1a transcript of the ERG1 gene. Using isoform-specific ERG1 antibodies, we have new evidence that subunits encoded by an alternate transcript, ERG1b, are also expressed in rat, canine, and human heart. The ERG1a and -1b subunits associate in vivo where they localize to the T tubules of ventricular myocytes. These data indicate native ventricular IKr channels are heteromers containing two alpha subunit types, ERG1a and -1b. The hERG1b-specific exon thus represents a novel target to screen for mutations causing type 2 long QT syndrome. These findings also suggest phenotypic analyses of existing type 2 long QT syndrome mutations, especially those exclusive to the hERG1a amino terminus, should be carried out in systems expressing both subunits.