AbstractIntroductionThe aim of this study was to describe and illustrate the technique of performing interatrial septum biopsy and to demonstrate its use for direct histological substrate characterization in atrial fibrillation (AF).Methods and ResultsBiopsies were performed in four patients who underwent AF catheter ablation. Bipal 7 bioptome was directed through a steerable sheath directly onto the septum. Fluoroscopic views as well as echocardiography‐guided techniques were utilized to confirm that the tip was oriented towards the interatrial septum. The bioptome was then placed on the right atrial (RA) septum and maneuvered to obtain the specimens (at least 1 mm in size) from the posterior septal region of the RA, adjacent to the fossa ovalis. Bioptome placement and sample acquisition were successful in all patients at the first attempt. No patient developed any minor or major complications during the procedure and hospital stay. All the biopsy specimens had proper qualities for histological assessments and revealed a variety of pathologies including fibrosis, inflammation, and fatty infiltration.ConclusionAtrial septum biopsies could be safely performed guided by fluoroscopy and transesophageal echocardiography. The obtained specimens allowed for a detailed localized substrate characterization which is of great interest in AF.