Abnormal myocardial conduction results from decreased gap junction (GJ) conductance (Gj). In particular phosphorylation of the GJ protein connexin 43 (Cx43) at serine-368 (pS368) by protein kinase C (PKC) is associated with reduced Gj. However, S368 phosphorylation only occurs after dephosphorylation of a neighbouring gatekeeper site, pS365. The protein phosphatase (PP) targeting pS365 is unknown. We aimed to: 1) assess the role of the Ca2+-dependent PP, calcinuerin (Cn) on Gj and protein levels of pS365 and pS368 when [Ca+2]i was raised; 2) examine if Cn acts directly or indirectly via another phosphatase, PP1, and its associated protein-inhibitor-1(I1).Male Dunkin-Hartley guinea-pigs were euthanised and left ventricular papillary muscles (LVPM) dissected in Tyrode’s solution (Na=147.4 mM). With an oil-gap technique, LVPM intracellular impedance was measured and Gj estimated in control or low-Na solution (Na=29.4 mM, to increase [Ca2+]i) and in the absence/presence of Cn inhibitors cyclosporine-A (CysA, 5 μM) or Cn autoinhibitory peptide (CAIP, 50 μM), or the PP1 inhibitor, tautomycin (TTM, 5 nM). Western blotting of tissue lysate measured: 1) total Cx43 (T-Cx43); 2) pS365 and pS368 – normalised to T-Cx43 which did not change in any intervention; 3) total I1 (T-I1) and pThr35-I1, both normalised to GAPDH. Values are mean±SEM, compared by ANOVA, the null hypothesis rejected at p<0.05.Gj was decreased in low-Na (59.8±3.6; n=8; p<0.001) compared to control (=100%). This was reversed by CysA (85.5±5.5%; n=8; p<0.001) or CAIP (109.8±23.1%; n=3; p<0.001), which alone had no effect. There was a significant decrease of pS365 in low-Na, compared to control (0.57±0.02 vs 0.95±0.03; n=3; p<0.05). This decrease was reversed by CysA (0.72±0.06, n=3; p<0.001) and CAIP (0.87±0.02, n=3; p<0.001). Whereas, there was a significant increase of pS368 in low-Na compared to control (0.9±0.03 vs 0.08±0.02; n=6; p<0.001) which in turn was reversed by CysA (0.15±0.9, n=6; p<0.001) and CAIP (0.13±0.03; n=6; p<0.001). The role of Cn in modulating pThr35-I1 was tested. T-I1 levels were similar in all interventions. There was a significant decrease of pTh35-I1 in low-Na compared to control (0.25±0.02 vs 0.83±0.00; n=6; p< 0.001) – reversed by CysA (0.37±0.02, n=6; p<0.05) and CAIP (0.71±0.08, n=6; p<0.001). These data suggest Cn dephosphorylated pThr35-I1 and activated PP1. This was further tested using TTM which showed similar effects to Cn inhibitors in low-Na by reversing the decrease of Gj and alterations to pS365 and pS368 as described above. Raised [Ca2+]i in ventricular myocardium is associated with a Cn-sensitive decrease of Gj by dephosphorylating pS365-Cx43, leading to enhanced S368 phosphorylation. Moreover, pS365-Cx43 is the target for two PPs, Cn and PP1: the latter activated by a Cn-dependent pathway.