Introduction: We have evidence that rapid cardiac pacing preserves gap junction (GJ) function by preventing the structural alterations that occur during ischaemia, 24h later; a mechanism which play a role the pacing-induced delayed protection against arrhythmias. Purpose: The present study has now examined whether closing of GJs prior to ischaemia with carbenoxolone (CBX) would attenuate the beneficial effects of pacing. Methods: Under light pentobarbitone anaesthesia 22 dogs were subjected to rapid right ventricular pacing (4x5 min, at a rate of 240 beats/min) and, 24 hours later, to a 30 min occlusion of the left anterior descending coronary artery (LAD). In 7 and 5 of these paced dogs CBX was infused locally into a small branch of the LAD in doses of 50 and 100 μM, respectively. The infusion was started 20 min prior to and maintained over the entire occlusion period. Another group of dogs (n=10) were instrumented but not paced and only subjected to occlusion. These dogs served as controls. At the end of the occlusion period, myocardial tissue samples were taken for the measurement of Cx43 phosphorylation, gap junction permeability, and immunofluorescence analysis of the intercalated discs (ID). Results: Compared with controls, cardiac pacing prevented the ischaemia-induced reduction in GJ permeability and the marked dephosphorylation of Cx43. Pacing also preserved the structural integrity of ID. CBX in both doses reversed the pacing-induced protection; i.e. the permeability of GJs was again decreased and the disruption of ID increased. CBX, however, did not affect the phosphorylation of Cx43. Conclusion: Our results that the inhibition of GJs with CBX prior to an ischaemic insult attenuates the protective effects of cardiac pacing confirm the role of GJs in the delayed phase of the pacing-induced protection.