Objective: We tested the hypothesis that structural remodeling of cellular connections, alterations in the expression of connexins (Cx), and an increase in fibrosis represent anatomic substrates of atrial fibrillation (AF). Methods: In 31 patients with AF undergoing a Maze procedure and 22 patients in sinus rhythm (SR), biopsies were taken intraoperatively from the right atrial (RA) free wall and appendages and investigated with immunoconfocal and electron microscopy. Results: All patients with AF exhibited a concomitant lateralization of gap junctional proteins Cx43 and Cx40, and N-cadherin (the major mechanical junction protein), instead of being confined to the intercalated discs, as observed in SR. These results were confirmed by quantitative immunoconfocal analysis and electron microscopy. Among diverse junctional proteins, in AF, Cx40 was markedly heterogenous in distribution. As compared with the SR group, Cx43 was significantly decreased in AF by 57% in RA appendages and by 56% in RA free wall. Cx40 was reduced by 54% in appendages, but had a tendency to be increased in the RA free wall. Collagen I was significantly higher in AF than in SR by 48% in RA appendages and by 69% in the RA free wall tissues. Conclusions: The structural correlate of AF comprises extensive concomitant remodeling of mechanical and electrical junctions, reduction of Cx43, heterogenous distribution of Cx40 in terms of different amounts of Cx40 in different RA tissues or in spatially adjacent regions of atrial myocardium. These changes, together with augmentation of fibrosis, may underlie localized conduction abnormalities and contribute to initiation and self-perpetuation of re-entry pathways and AF.