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Introduction Epicardial catheter ablation is increasingly used to treat arrhythmias with an epicardial component. Nevertheless, percutaneous epicardial access remains associated with a significant risk of major complications. Developing a technology capable of confirming proper placement within the pericardial space could decrease complication rates. The purpose of this study was to examine differences in bioimpedance among the pericardial space, anterior mediastinum, and right ventricle. Methods An ovine model (n = 3) was used in this proof-of-concept study. A decapolar catheter was used to collect bipolar impedance readings; data were collected between each of five electrode pairs of varying distances. Data were collected from three test regions: the pericardial space, anterior mediastinum, and right ventricle. A control region in the inferior vena cava was used to normalize the data from the test regions. Analysis of variance was used to test for differences among regions. Results A total of 10 impedance values were collected in each animal between each of the five electrode pairs in the three test regions (n = 340) and the control region (n = 145). The average normalized impedance values were significantly different among the pericardial space (1.760 ± 0.370), anterior mediastinum (3.209 ± 0.227), and right ventricle (1.024 ± 0.207; P < 0.0001). In post hoc testing, the differences between each pair of regions were significant, as well (P < 0.001 for all). Conclusion Impedance values are significantly different among these three anatomical compartments. Therefore, impedance can be potentially used as a means to guide percutaneous epicardial access. [ABSTRACT FROM AUTHOR]