BACKGROUND Sustained atrial fibrillation (AF) causes alterations in atrial electrical and structural properties. Conflicting data regarding the structural remodeling of the gap junction proteins connexin (Cx) 40 and 43 in human and animal studies exists. We investigated the amount and distribution of Cx40 and Cx43 in three subtypes of AF. METHODS In 50 patients undergoing coronary artery bypass graft and/or mitral or aortic valve surgery, right atrial appendages were taken and examined with immunoconfocal microscopy. Retrospectively, four groups were built: (1) sinus rhythm pre- and postoperative (SR, n=20), (2) intermittent AF, but SR prior to surgery (intAF, n=6), (3) postoperative AF (popAF, n=12), and (4) persistent AF, at least 3 month prior to surgery (persAF, n=12). We analyzed the amount of Cx40 and Cx43 and the degree of fibrosis in three randomly selected areas of each sample. RESULTS As compared with SR, the amount of Cx40 was significantly reduced by 53% in persAF. The distribution pattern of Cx40 was heterogeneous in patients with SR, intAF, and popAF, whereas patients with persAF showed similar densities of Cx40 in the three examined areas. We found no significant difference in the amount of Cx43 between the four groups. The distribution pattern of Cx43 was heterogeneous in all four groups. The Cx40/Cx43 ratio was significantly reduced in patients with popAF and persAF by 51% and 53%, respectively. No difference was seen in the degree of fibrosis between the four groups. CONCLUSIONS In this study, sustained AF leads to a reduction in the amount of Cx40. Together with a specific Cx40/Cx43 ratio, this may contribute to localized conduction abnormalities, facilitating the self-perpetuation of re-entry pathways in AF. In the time course of structural atrial remodeling these changes seem to be earlier than a concomitantly developing fibrosis.