In contrast to the pulmonary veins as a well-accepted site for triggering paroxysmal atrial fibrillation/flutter (AF/AFL), little is known about the intrinsic basis for reentrant excitation in persistent AF/AFL. To explore histoanatomic substrates for reentrant AF/AFL in atria, we conducted functional and histoanatomic studies of the whole atria of rats by combining optical imaging of impulse propagation with histologic examination. After sufficient general anaesthesia applied to male Wistar rats (n=19, 11-14 weeks, 290-430 g) by single intra-abdominal injection of sodium pentobarbitone (19 mg/100g), hearts with bilateral lungs were excised and were quickly perfused in a Langendorff manner. Spatiotemporal patterns of impulse propagation were visualized optically on the posterior surface of the atria stained with di-4ANEPPS at 32 °C with cytocholasin D (4 μmol/L). AF/AFL was evoked by single extra stimulation (S2) after 5-Hz consecutive pacing (S1) at the right atrium (RA). After the optical imaging we quantified regional differences in 3 histological determinants for conductivity, i.e., myocardial density, myofibre orientation, and distribution of gap junctional (connexin 43) proteins (n = 10). The myocardial density was evaluated by Masson’s trichrome staining, and other two parameters were assessed by confocal immunohistochemistry. Quantitative data (mean±SD) were analysed by paired t-test or one-factor ANOVA. Burst (S1-S2) pacing at the RA provoked AF/AFL in 15 of 19 hearts, and most cases developed by organized reentrant excitation through the coronary sinus (CS) and left atrium (LA) roof, with non-organized irregular propagation in 3 cases. The reentrant circuit developed along 2 directions of propagation in the LA: a slower one at the LA roof and a faster one along the CS, with conduction velocities of 42.4±16.6 cm/s and 53.3±9.2 cm/s, respectively (p<0.005). Upon S2 stimulus the impulse at the LA roof propagated retrogradely from the CS, resulting in reentrant propagation that mediated through the CS and the LA roof. Histologic quantification revealed significantly lower myocardial density in the posterior LA and the septum than in the remainder of the atria, suggesting a possible electrical obstacle. Moreover, myocytes in the LA roof were significantly of lower density (p<0.005) and arranged more randomly in the direction of conduction than those in the CS. Connexin 43 plaques were distributed randomly over the entire cell membrane in the LA roof (slower pathway), whereas those in the CS (faster pathway) were located in orderly fashion. We identified an intrinsic histoanatomic basis in the LA, especially the posterior LA, septum and LA roof, for formation of reentrant propagation in the atria. These regional heterogeneities observed in the LA would constitute a pro-reentrant substrate for perpetuating AF/AFL.