The pentamidine analog PA-6 was developed as a specific inward rectifier potassium current (IK1) antagonist, because established inhibitors either lack specificity or have side effects that prohibit their use in vivo. We previously demonstrated that BaCl2, an established IK1inhibitor, could prolong action potential duration (APD) and increase cardiac conduction velocity (CV). However, few studies have addressed whether targeted IK1inhibition 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 (APD90). Ventricular APD90was 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 APD90during hypokalemia (2 mmol/L [K+]o), although to a lesser degree than observed at 4.56 mmol/L [K+]oIn 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 APD90to a lesser degree, but profoundly increased CV Thus, in intact guinea pig hearts, the electrophysiologic effects of the IK1inhibitor, PA-6, are [K+]o-dependent.