Abstract Funding Acknowledgements Type of funding sources: None. Background Unpredictable lesion formation is a major limiting factor for radiofrequency catheter ablation for atrial fibrillation (AF). Ablation index (AI) emerged as a novel parameter to monitor lesion development and potentially improve clinical outcomes. Additional evidence is needed to support its use. Purpose We aimed at evaluating the relationship between AI and other lesion indicators and the release of myocardial-specific biomarkers following AF ablation. Methods Forty-six patients with similar baseline characteristics underwent a first-time radiofrequency ablation for AF and were prospectively enrolled in this study. Pulmonary vein isolation was performed by six experienced electrophysiologists with a point-by-point approach, guided by strict Visitag criteria and consistent AI target values. Myocardial-specific biomarkers troponin T and creatine kinase myocardial band were measured after 6 (TnT6 and CKMB6) and 20 hours (TnT20 and CKMB20) following sheath removal. Ablation duration, impedance drop (ID), force-time integral (FTI) and AI were registered automatically and analyzed off-line. Since biomarkers release reflect the total amount of myocardial injury, our independent variables consisted of total ablation duration, total ID, total FTI and total AI. Results 97% of total ablation duration fulfilled pre-specified Visitag criteria. Mean application duration was 20.0 ± 3.5 s, mean contact force was 15.9 ± 4.2 g, mean maximum power was 35.3 ± 1.5 W. Troponin T release was 985 ± 495 ng/L and 1038 ± 461 ng/L (p = ns) while CKMB release was 7.3 ± 2.7 mcg/L and 6.5 ± 2.1 mcg/L (p < 0.001) at 6 and 20 hours, respectively. Ablation duration, ID, FTI and AI were all significantly correlated with the release of myocardial-specific biomarkers both at 6 and 20 hours (all correlations significant at p < 0.01 level, Fig. 1). Ablation index showed the highest degree of correlation with TnT6, TnT20, CKMB6 and CKMB20 (Pearson`s R 0.69, 0.69, 0.58, 0.64 respectively, p < 0.001). Multiple regression analysis demonstrated that AI had the strongest association with TnT6, TnT20, CKMB6 and CKMB20 (beta 0.43, 0.71, 0.41 and 0.43, respectively). Conclusion Ablation index appears as the strongest lesion size indicator as measured by the release of myocardial-specific biomarkers following radiofrequency catheter ablation for AF. Abstract Figure. Scatter plots and correlations