The G-protein-regulated, inwardly rectifying K+ (GIRK) channels are critical for functions as diverse as heart rate modulation and neuronal post-synaptic inhibition. GIRK channels are distributed predominantly throughout the heart, brain, and pancreas. In recent years, GIRK channels have received a great deal of attention for their direct G-protein betagamma (Gbetagamma) regulation. Native cardiac IKACh is composed of GIRK1 and GIRK4 subunits (Krapivinsky, G., Gordon, E. A., Wickman, K. A., Velimirovic, B., Krapivinsky, L., and Clapham, D. E. (1995) Nature 374, 135-141). Here, we examine the quaternary structure of IKACh using a variety of complementary approaches. Complete cross-linking of purified atrial IKACh protein formed a single adduct with a total molecular weight that was most consistent with a tetramer. In addition, partial cross-linking of purified IKACh produced subsets of molecular weights consistent with monomers, dimers, trimers, and tetramers. Within the presumed protein dimers, GIRK1-GIRK1 and GIRK4-GIRK4 adducts were formed, indicating that the tetramer was composed of two GIRK1 and two GIRK4 subunits. This 1:1 GIRK1 to GIRK4 stoichiometry was confirmed by two independent means, including densitometry of both silver-stained and Western-blotted native atrial IKACh. Similar experimental results could potentially be obtained if GIRK1 and GIRK4 subunits assembled randomly as 2:2 and equally sized populations of 3:1 and 1:3 tetramers. We also show that GIRK subunits may form homotetramers in expression systems, although the evidence to date suggests that GIRK1 homotetramers are not functional. We conclude that the inwardly rectifying atrial K+ channel, IKACh, a prototypical GIRK channel, is a heterotetramer and is most likely composed of two GIRK1 subunits and two GIRK4 subunits.