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Aims Catheter ablation of ventricular tachycardia (VT) is an effective treatment in patients with structural heart disease (SHD) and recurrent arrhythmias. However, the procedure is associated with the risk of complications, including both manifest and asymptomatic cerebral thromboembolic events. We hypothesized that periprocedural asymptomatic brain injury (ABI) can be reduced by using transseptal instead of the retrograde access route to the left ventricle (LV). Methods and results Consecutive patients undergoing VT ablation for SHD were randomized 1:1 to either retrograde or transseptal LV access. All patients underwent radiofrequency ablation in conscious sedation with the use of an irrigated tip catheter. The degree of brain damage was evaluated by serum level of biomarker S100B. Significant ABI was defined as a post-ablation relative increase of S100B level >30%. A total of 144 patients (66 ± 9 years; 14 females; 90% coronary artery disease; LV ejection fraction: 30 ± 8%) were enrolled and 72 were allocated to each study groups. Symptomatic neurological complication of the procedure was not observed in any subject. A significant ABI was detected in 19.4% of patients. It was more commonly observed in subjects randomized to retrograde vs. transseptal LV access (26.4% vs. 12.5%, P = 0.04). In a multivariate analysis, only retrograde LV access and advanced age were independent determinants of significant ABI. Conclusion Significant ABI after ablation of VT in patients with SHD can be detected in one-fifth of subjects. Retrograde access to LV is associated with a two-fold higher probability of significant ABI.