Cardiac arrhythmias are significantly initiated by abnormal myocardial conduction, in part caused by raised intracellular resistivity, Ri. In turn, Ri is a function of sarcoplasmic resistivity, Rc, and gap junction (GJ) resistivity, Rj. GJs are composed of connexin (Cx) proteins and in many cardiac arrhythmias, such as atrial fibrillation, changes in GJ electrical properties can be linked to alteration in the phosphorylation state of Cx and/or raised [Ca2+]i. A key intracellular signalling pathway affected by raised [Ca2+]i is the activation of Ca2+/calmodulin dependent, serine/threonine phosphatase; calcineurin (CaN). The objectives of this study were to: 1) determine if raised [Ca2+]i in atrial myocardium altered Rj and if this was reversible with the CaN inhibitor cyclosporin A (CsA); and 2) measure the effect of increased [Ca2+]i on the phosphorylation state of Cx protein, Cx43. Male, Dunkin-Hartley guinea-pigs were sacrificed, hearts removed and transferred to Tyrode’s solution (total [Na+]=147.4mM; 24 mM NaHCO3, 5%CO2, 37°C, pH 7.4). Using an oil-gap technique, tissue impedance (Z) was measured to derive Rj and Rc from Z values over a range of frequencies (0.02-100 kHz). These values were calculated from atrial appendage strips bathed in control and low Na+ Tyrode’s solutions (29.4 mM Na+, NaCl replaced by TRIS HCl, pH 7.4; used to increase [Ca2+]i) in the presence and absence of 5 µm CsA. The protein expression of total Cx43 (T-Cx43) and (de)phosphorylation of the serine 368 residue of Cx43 (dephos-S368 and pS368) of tissue homogenates were determined using western blotting. Dephos-S368 and pS368 were normalised to corresponding T-Cx43. Data are means ± SEM, compared by Mann Whitney or ANOVA, the null hypothesis rejected at p<0.05. Rj was significantly increased by low Na+ (383 ± 40 Ω.cm) when compared to control (209 ± 26 Ω.cm; n=6; p<0.0001). This effect was reversed in the presence of 5 µm CsA (250 ±27 Ω.cm; n=6; p<0.0001), which alone had no effect (181 ± 17 Ω.cm). Rc did not change significantly during any intervention. Western blots did not show any difference in T-Cx43 protein expression between control and low Na+. However, there was a significant increase in the normalised pS368 in low Na+ tissue compared to control tissue (pS368; Control, 59.6 ± 25.2, low Na+ 115.1 ± 17.1; n=3; p< 0.05). Raised [Ca2+]i in atrial myocardium is associated with an increased Rj, as well as greater phosphorylation of the S368 residue of Cx43; the increase of Rj was ameliorated by the CaN inhibitor CsA. Phosphorylation of S368 of Cx43 is associated with a lower conductance state of gap junctions. We propose that CaN increases GJ resistivity by enhancing the phosphorylation of the S368 residue of Cx43 and decreasing GJ conductance.