We recently characterized two distinct mechanisms by which the polyamine spermine blocks Kir2.1 channels: (1) by reduction of negative surface charges in the cytoplasmicpore, thereby reducing single‐channel conductance, and (2) by direct open channel transmembrane pore block. The extent to which the surface charge reduction component is mediated by passive surface charge screening versus binding of polyamines to these charges, as well as the extent to which the surface charge reduction and pore block mechanisms are synergistic, versus simply additive, was not established. To address these issues, macroscopic currents were recorded from inside‐out giant patches from Xenopus oocytes and from single‐channel currents from COS7 cells expressing wild‐type and mutant Kir2.1 channels, during exposure to polyamines of varying length and charge. The surface charge reduction component was decreased when polyamine charge (at constant length) was decreased from 4 (spermine) to 2 (diamine 10, DA10). Moreover, the surface charge reduction component of block involved more than passive surface charge screening and required binding of polyamines to the cytoplasmic pore, since it was eliminated when polyamine length was shortened below six alkyl groups. Loss of surface charge reduction also dramatically affected open channel pore block. The latter consisted of two subcomponents with fast and slow kinetics, respectively. The slow subcomponent decreased as blocker length decreased (DA10, DA8 and DA6), whereas the fast subcomponent was sensitive to blocker charge (spermine vs. DA10). Neutralization of E224 and E299, which eliminated the surface charge reduction component of block, also eliminated the fast subcomponent of pore block. Neutralization of D172 had no effect on the surface charge reduction component, but weakened both of the subcomponents of pore block. These findings can be accounted for by a model in which the negative charges at E224, E299 and D172 act in a concerted manner to coordinate the surface charge reduction and open channel components of polyamine block. In this model, the binding of polyamines to surface charges E224 and E299 pre‐positions them in the cytoplasmic pore in a manner that directly facilitates their entry and exit from a transmembrane pore‐occluding site involving D172. A molecular model using the recently reported 1.8 Å resolution structure of the inward‐rectifier cytoplasmic pore, adapted to Kir2.1, is consistent with longer polyamines binding at their positively charged ends to the E224 and E299 positions in the same subunit, potentially accommodating four polyamine molecules per channel.