Connexins are known to play an essential role in the ischemic preconditioning (IP) of the heart, their functional role in this process, however, has not yet been clearly defined. For this reason anesthetized rats were subjected to regional myocardial ischemia, either with or without preceding IP and/or following reperfusion. In freeze sections of the hearts fluorescence immunohistochemical staining for connexin43 (Cx43) was performed. In contrast to undisturbed zones, tissue which had been subjected to ischemia revealed Cx43 immunostaining not only in the gap junctions but also in a conspicuous pattern in the free cellular membranes of the myocytes. In myocardium which was solely exposed to IP, the ratio of immunofluorescence intensity in the free cell membrane to the cell's interior was 1.22 ± 0.04 (non-ischemia-exposed area: 1.04 ± 0.01). When a 15 min or 45 min permanent ischemia followed IP, the effect became more evident (ratios 1.31 ± 0.03 and 1.46 ± 0.03, respectively) and proved to be significantly greater than in the corresponding non-IP-pretreated groups (1.16 ± 0.03 and 1.30 ± 0.03, p<0.01, respectively). Reperfusion led to an overall weakening of fluorescence intensities and a disappearance of the observed IP-specific differences. We conclude that IP initiates a redistribution of Cx43 from its natural position in the gap junctions towards the free plasma membrane, thereby improving the chance of the cells' survival during the subsequent phase of prolonged ischemia by a yet unknown, supposedly gap-junction-independent mechanism.