Action potential conduction velocity in myocardium is a function of gap junction (GJ) electrical conductance [1]. GJ conductance (Gj) itself depends on the phosphorylation state of GJ proteins, connexins (Cx). We have shown that during elevated intracellular Ca2+ concentration ([Ca2+]i) phosphatases mediate a decrease of Gj in the left atrium (LA). Consequently we investigated the role of serine threonine protein phosphatases (PPs) on Cx phosphorylation, in particular the Ca2+-dependent PP, calcineurin, and the Ca2+-independent PPs, PP1, PP2A. Male, Dunkin-Hartley guinea pigs were euthanised and the LA dissected. Freshly isolated atrial cardiomyocytes were used to measure [Ca2+]i using Fura-2 AM in control (Na=149.4 mM) or low-Na solution (Na=29.4 mM). Samples used for western blots were perfused in control or low-Na solution in the absence/presence of inhibitors for: calcineurin (cyclosporin-A (CysA; 5 µM; n=4) or calcineurin auto-inhibitory peptide (CAIP; 50 µM, n=4)), PP1 (tautomycin (TTM; 5 nM; n=4)) and PP2A (fostriecin; FST; 100 nM; n=4). Western blots were used to assess protein expression of total-Cx43 (T-Cx43), T-Cx40 and Cx43 phosphorylation at Ser368 (Cx43-pSer368) and Ser365 (Cx43-pSer365). Immunoprecipitated T-Cx40 was also probed for serine and threonine phosphorylation. Values are integrated density normalised to GAPDH or T-Cx and shown as mean±SEM. Differences between sets were tested by ANOVA; the null hypothesis was rejected at p<0.05. Low-Na solution significantly and reversibly increased [Ca2+]i. T-Cx43 and T-Cx40 were unchanged throughout all interventions. During low-Na exposure, phosphorylation at Cx43-pSer368 was significantly increased (control 0.05±0.01 vs low-Na 1.09±0.04; p<0.001); this was reduced by inhibition of calcineurin (low-Na+CAIP 0.03±0.01; p<0.001) and PP2A (low-Na+FST 0.58±0.05; p<0.001), but not PP1 (low-Na+TTM 1.01±0.03). In parallel, Cx43-pSer365, which is phosphorylated under control, was diminished during low-Na solution; this was attenuated during FST treatment (control 0.50±0.02 vs low-Na 0.38±0.02 vs low-Na+FST 0.47±0.03; n=3; p<0.05). Phosphorylation of T-Cx40 at serine and threonine residues was significantly increased in low-Na solution (control 0.09±0.04 vs low-Na 1.06±0.06; p<0.001). This was partially reduced by CysA (0.47±0.04; p<0.01) and FST (0.61±0.04; p<0.01) and returned to control levels during exposure to CAIP (0.11±0.03; p<0.01) but TTM had no effect (1.08±0.02). This study showed that Cx43-Ser368 phosphorylation by protein kinase C is mediated by calcineurin and PP2A via initial dephosphorylation of Cx43-Ser365. Furthermore we showed that during raised [Ca2+]i Cx40 phosphorylation is increased at serine and threonine residues and this is also mediated solely by calcineurin with partial involvement of PP2A. There was no evidence of a role for PP1 in phosphorylating Cx40 in the (LA).