INTRODUCTION: Fibrotic heart is known to have high incidence of atrial fibrillation (AF), but underlying mechanisms are not clearly defined. To test the hypothesis that atrial fibrosis promotes early afterdepolarization (EAD) and triggered activity. METHODS: MHC-TGFcys³³ser transgenic (Tx) mice develop atrial, but not ventricular, fibrosis due to elevated levels of TGF-棺1. We optically mapped changes in membrane potential (V_m) and intracellular calcium (Ca_i) in superfused atria isolated from 18 Tx mice and 18 wild type (Wt) littermates. RESULTS: At baseline, atrial fibrillation (AF) was induced by burst stimulation in Tx atria (61%) but not in Wt atria (0%, p<0.001). However, AF with multiple reentrant wavefronts could be induced in both Tx (100%) and Wt (71%, p=NS) atria during carbachol (1-3 µM) superfusion. Repetitive early afterdepolarizations (EADs) and triggered activity were observed following rapid atrial pacing in Tx (58%) but not in Wt atria (0%, p=0.001). Most EADs occurred in the presence of shortened action potential duration and prolonged Cai transients. Only Tx atria developed spontaneously recurrent AF during carbachol superfusion (39 vs. 0%, p=0.008). The recurrent AF was initiated by EAD and repetitive triggered activities with focal discharge patterns. We were able to map optically 4 episodes of spontaneous AF reinitiation. All first and second beats of spontaneous AF originated from the right atrium (4/4, 100%). Interestingly, fibrosis was also more severe in right atria of Tx mice. Tx RA showed more fibrosis than Tx LA, Wt RA and Wt LA (27.1 짹 2.6%, 18.7 짹 2.6%, 3.6 짹 0.6% and 4.7 짹 0.5%, respectively, all p<0.05 compared to Tx RA). Ryanodine (3 µM) and thapsigargin (1 µM) prevented the persistent Ca_i elevation during late phase 3 and early phase 4 of the action potential, and inhibited spontaneous re-initiation of AF in all 7 Tx atria tested. CONCLUSION: TGF-棺1 over-expression was accompanied by increased vulnerability to EADs, triggered activity and spontaneous recurrences of AF. Pharmacological inhibition of sarcoplasmic reticulum Ca²⁺ release effectively suppressed spontaneous AF recurrence. Our results support a role of Ca²⁺-dependent triggered activity in spontaneous AF recurrence in this model.