Congenital heart defects (CHD) are common in Down syndrome (DS, trisomy 21). Recently, cardiac sympathetic-parasympathetic imbalance has also been documented in DS adults free of any CHD. The KCNJ6 gene located on human chromosome 21 encodes for the Kir3.2/GIRK2 protein subunits of G protein regulated K⁺ (K_G) channels and could contribute to this altered cardiac regulation. To elucidate the role of its overexpression, we used homozygous transgenic (Tg⁺⁺) mice carrying copies of human KCNJ6. These mice showed human Kir3.2 m-RNA expression in the heart and a 2.5 fold increased translation in the atria. Phenotypic alterations were assessed by recording electrocardiogram of urethane anesthetized mice. Chronotropic responses to direct (carbachol) and indirect (methoxamine) muscarinic stimulation were enhanced in Tg⁺⁺ mice with respect to wild type (WT) mice. Alternating periods of slow and fast rhythm induced by CCPA (2-chloro-N-cyclopentyl-adenosine) were amplified in Tg⁺⁺ mice, resulting in a reduced negative chronotropic effect. These drugs reduced the atrial P wave amplitude and area. P wave variations induced by methoxamine and CCPA were respectively increased and reduced in the Tg⁺⁺ mice while PR interval and ventricular wave showed no difference between Tg⁺⁺ and WT. These results indicate that Tg⁺⁺ mice incorporating the human KCNJ6 exhibit altered Kir3.2 expression and responses to drugs which would activate K_G channels. Moreover, these altered expression and responses are limited to sino-atrial node and atria that normally express large amounts of K_G channels. These data suggest that KCNJ6 could play an important role in altered cardiac regulation in DS patients.