The pentamidine analog PA-6 was developed as a specific inward rectifier potassium current (I-K1) antagonist, because established inhibitors either lack specificity or have side effects that prohibit their use in vivo. We previously demonstrated that BaCl2, an established I-K1 inhibitor, could prolong action potential duration (APD) and increase cardiac conduction velocity (CV). However, few studies have addressed whether targeted I-K1 inhibition similarly affects ventricular electrophysiology. The aim of this study was to determine the effects of PA-6 on cardiac repolarization and conduction in Langendorff-perfused guinea pig hearts. PA-6 (200 nm) or vehicle was perfused into ex-vivo guinea pig hearts for 60 min. Hearts were optically mapped with di-4-ANEPPS to quantify CV and APD at 90% repolarization (APD(90)). Ventricular APD90 was significantly prolonged in hearts treated with PA-6 (115 +/- 2% of baseline; P < 0.05), but not vehicle (105 +/- 2% of baseline). PA-6 slightly, but significantly, increased transverse CV by 7%. PA-6 significantly prolonged APD90 during hypokalemia (2 mmol/L K+), although to a lesser degree than observed at 4.56 mmol/L K+. In contrast, the effect of PA-6 on CV was more pronounced during hypokalemia, where transverse CV with PA-6 (24 +/- 2 cm/sec) was significantly faster than with vehicle (13 +/- 3 cm/sec, P < 0.05). These results show that under normokalemic conditions, PA-6 significantly prolonged APD90, whereas its effect on CV was modest. During hypokalemia, PA-6 prolonged APD90 to a lesser degree, but profoundly increased CV. Thus, in intact guinea pig hearts, the electrophysiologic effects of the I-K1 inhibitor, PA-6, are K+-dependent.