Previous studies have shown that cardiac inward rectifier potassium current (I_K1) channels are heteromers of distinct Kir2 subunits and suggested that species- and tissue-dependent expression of these subunits may underlie variability of I_K1. In this study, we investigated the contribution of the slowly activating Kir2.3 subunit and free intracellular polyamines (PAs) to variability of I_K1 in the mouse heart. The kinetics of activation was measured in Kir2 concatemeric tetramers with known subunit stoichiometry. Inclusion of only one Kir2.3 subunit to a Kir2.1 channel led to an approximate threefold slowing of activation kinetics, with greater slowing on subsequent additions of Kir2.3 subunits. Activation kinetics of I_K1 in both ventricles and both atria was found to correspond to fast-activating Kir2.1/Kir2.2 channels, suggesting no major contribution of Kir2.3 subunits. In contrast, I_K1 displayed significant variation in both the current density and inward rectification, suggesting involvement of intracellular PAs. The total levels of PAs were similar across the mouse heart. Measurements of the free intracellular PAs in isolated myocytes, using transgenically expressed Kir2.1 channels as PA sensors, revealed “microheterogeneity” of I_K1 rectification as well as lower levels of free PAs in atrial myocytes compared with ventricular cells. These findings provide a quantitative explanation for the regional heterogeneity of I_K1.