Cellular and ionic mechanisms of pentamidine-mediated acute and chronic inhibition of cardiac inward rectifier current were examined (IK1). Pentamidine is an antiprotozoal drug widely used in treatment of Leishmaniasis, Human African trypanosomiasis and opportunistic protozoal infections resulting in candidiasis and pneumonia. Pentamidine treatment may cause sudden cardiac death by provoking cardiac arrhythmias associated with QTc prolongation and U-wave alterations. We hypothesize that pentamidine reduces Kir2.1 carried IK1 current. Patch clamp measurements of IK1 on adult canine ventricular cardiomyocytes, Kir2.1-HEK293 cells and Kir2.x inside-out patches were performed. Kir2.1 protein expression was measured by western blotting. Pentamidine binding simulation was performed using a computer model. Pentamidine decreases IK1 in cardiomyocytes (10 mM) and Kir2.1-HEK293 (1 and 10 mM) after 24 h incubation. In Kir2.1-HEK293, Kir2.1 protein expression decreases as a function of pentamidine concentration (1–10 mM). The effect is not acute, but develops after chronic exposure to the agent (16–72 h). Inhibition of lysosomal, but not proteasomal, degradation partially rescues Kir2.1 protein expression levels. When applied from the cytoplasmic side, pentamidine block of IK1 is acute (IC50=0.17 mM), and depends on the negatively charged amino acids E224, D259 and E299 in the cytoplasmic pore region of Kir2.1, but is independent of the presence of polyamines or magnesium. Kir2.2 and Kir2.3 based IK1 is also sensitive to pentamidine blockade. Molecular computer modeling confirms stable binding of pentamidine in the cytoplasmic pore region with residues E224, D259 and E299. Pentamidine inhibits cardiac IK1 by a dual mechanism. There are a chronic (lowering Kir2.1 protein expression levels) and an acute component (direct Kir2.x ion channel block from the cytoplasmic side). Kir2.1 protein levels can be rescued by lysosomal inhibition.